Detection of oral microbial virulence factors

ABSTRACT

Methods for detecting and quantifying toxins present in the oral cavity. The methods may include providing a biological sample, providing reporter cells expressing one or more Toll like receptors, exposing the cells to the biological sample, measuring the EC50 value of the lipopolysaccharide on activation of a Toll like receptor, quantification of the lipopolysaccharide in the biological sample.

FIELD OF THE INVENTION

The invention relates to a method for detecting and quantifyingbacterial virulence factors in oral samples, and for identifying agentsfor detoxifying oral virulence factors and a method for determining theeffectiveness of detoxifying agents.

BACKGROUND OF THE INVENTION

The toxins of bacterial cell walls (Henkel et al, EXS. (2010) 100: 1-29)have been associated with health related issues, such as septic shock,fever and malaise (V. Liebers et al., Am J Ind Med. (2006) 49(6):474-91). Examples of Gram-negative cell wall toxins associated withhealth concerns are endotoxins, such as lipopolysaccharide (LPS),peptidogylcans, and fimbriae; and Gram-positive cell wall toxinsassociated with health concerns are lipoteichoic acid (LTA) andpeptidoglycans. There are many other bacterial toxins, such asenterotoxins and exotoxins, as reported in Henkel et al, EXS. 2010; 100:1-29. For the oral environment, the LPS and LTA seem to be the dominantdrivers of a bacterial induced immune response, or at least the bestcharacterized. The immune response mounted by the body in response tothese toxins depends on the origin of the toxin and the exposure historyof the individual to said toxin. The LPS is a component of Gram-negativebacteria that is different from strain to strain, as has beenillustrated with the differences in virulence of E. coli (Raetz andWhitfield Annu. Rev. Biochem (2002) 71:635-700). LPS is composed of alipid A fraction, core region, and may have an O-antigen. The Lipid Afraction's fatty acid composition has been shown to determine itsvirulence in response to its interaction with the Toll-like 4 (TLR4)receptor. The LTA has been linked to various inflammatory responses (Y.Yokoyama, et al., Acta Otolaryngol Suppl. (1996) 523:108-111) andassociated with Toll-like receptor 2 (TLR2) activation. It is widelybelieved that only the lysed bacteria liberate LPS that can initiate aninflammatory response (CA2323630). However, Zhang et al. showed thatgrowing bacteria secrete LPS at a level in proportion to their growthphase (H Zhang et al. (1998) Infection & Immunity, 66(11), 5196-5201).Therefore, even a small fraction of the plaque left on the teeth afterbrushing could seed the inflammatory cascade due to the release of LPSfrom the Gram negative bacteria present in the plaque.

Methods of detecting specific microbial species have been demonstratedin the art. In US Pub. No. 2012/019735A1, methods were proposed todistinguish disease-causing bacteria via spectrophotometric methods.Though they were able to show the presence of specific microbes, theirinvention would not allow the user to determine the virulence level of aspecific site. Further, their method requires the microbes to becultured in the lab in order to obtain a sufficient quantity of LTA orLPS for detection. Thus, their invention lacks the ability to detect thenon-culturable species present, nor would it allow for measures oftoxicity of biological samples.

In U.S. Pat. No. 5,175,089, the use of the Limulus amebocyte lysate(LAL) endotoxin (LPS) assay was applied to the determination of theamount of endotoxin in the periodontal pocket. Though they were able toshow overall amounts of endotoxin present, they lacked the ability todifferentiate diseased versus healthy endotoxin and they were unable toquantify the level of virulence of the endotoxin. Further, theirinvention limited them to the Gram-negative endotoxin, as the LTA is notdetectable via the LAL kit.

In US Pub. No. 2009/0047240, the chaperonin 10 (Cpn10) was used tomodulate the clustering of Cpn10 in a cell line (murine RAW264) withlabeled antibodies. Though they showed TLR-4, 7, and 9 reporter genes inan HEK cell line, their system would not allow for a more sensitive orlow level detection needed for microbial populations with weakeractivating LPS, since those genes were under the control of the NFkBbinding sites only (a minimal promoter). Their system lacks thesensitivity needed to differentiate biological systems with multiplemicrobial species and no dominant organism present. Further, theirsystem needs strong NFkB activators to overcome the weak promoter usedin their system, thus unable to pick up weaker TLR LPS agonists, such asLPS from Porphyromonas gingivalis. Additionally, their system lacked theability to detect TLR3 agonists, which would be deleterious to thecharacterization of an inflammatory disease, such as gingivitis.

US Pub. No. 2007/0160544 describes a method for determining orallydeleterious bacteria. Their method calls for contacting a gingival cellwith bacteria or a bacterial component and measuring an inflammatorymarker. According to US Pub. No. 2007/0160544, the presence of a markerindicates inflammation and the labeling of a bacterium as deleterious.Conversely, they say that the absence of a marker indicates thebacterium is not a problem. Though they cited Toll-like receptors, whichwere known in the art as part of the pathway to generate cytokines,their method would have only allowed for determining the presence of acytokine.

Since oral cells contain one or more of the receptors to which abacterial virulence factor would activate, screening on the individualreceptors requires the use of engineered cells, such as reporter cellscontaining the receptor gene of interest. What further complicates theuse of native oral cells, such as gingival cells, is that the expressionand activation of a receptor, such as a Toll-like receptor, is specificto the function of the cell. Gingival cells are less likely to respondto bacterial virulence factors, due to their constant contact withmicrobes in the dental plaque. Thus the need exists to have engineeredcells where a direct response can be measured via a reporter system.

In addition to quantifying the virulence of microbial components andbyproducts, there also exists a need for an in vitro screen of theinflammatory potential of organic and inorganic molecules, which wouldallow for pharmokinetic parameters to be determined.

SUMMARY OF THE INVENTION

A method of determining bacterial virulence in an oral cavity isprovided that includes providing a reporter cell expressing at least onetoll-like receptor; providing a sample of oral matter; combining thesample of oral matter and the reporter cell; and measuring the toll-likereceptor activation. The method may also include the additional steps ofproviding another sample of oral matter from an individual who used anoral care composition prior to providing another sample of oral matter;combining the another sample of oral matter and the reporter cell;measuring the toll-like receptor response; and comparing the toll-likereceptor response from the sample of oral matter and another sample oforal matter.

A method for determining the virulence of lipopolysaccharide comprisingproviding a lipopolysaccharide; providing a reporter cell expressing atleast one Toll-like receptor; combining the reporter cell with thelipopolysaccharide; measuring the toll-like receptor activation; andquantifying the lipopolysaccharide.

A method for determining the virulence of lipoteichoic acid comprisingproviding a lipoteichoic acid; providing a reporter cell expressing atleast one Toll-like receptor; combining the reporter cell with thelipoteichoic acid; measuring the toll-like receptor activation; andquantifying the lipoteichoic acid.

The present invention includes methods of improving and/or resolving thestate of gingival inflammation utilizing molecules, peptides, orproteins/enzymes that bind to, alter, or chemically modify bacterialvirulence factors and/or host response mechanisms. The methods provide ameans of quantifying the level of lipopolysaccharide of oral tissues;and utilizes TLR-4 and TLR-2 reporter cells line combined with thedetection LPS via a fluorescence assay, such as BODIPY TR cadaverine, orendotoxin detection assay, assay to assign potency and quantification ofLPS. The invention also includes assays and protocols which enablecommunication and demonstrations to consumers and dental professionalsutilizing the TLR reporter cells.

The present invention includes methods for determining the potency oflipopolysaccharide comprising: a) providing a lipopolysaccharide sample;b) providing reporter cells expressing one or more Toll-like receptors;c) exposing the cells to the lipopolysaccharide sample; d) measuring theEC50 value of the lipopolysaccharide on activation of a Toll-likereceptor; e) quantification of the lipopolysaccharide.

The present invention also includes isolating lipopolysaccharide from agrowth culture of Gram negative bacteria. A lipopolysaccharide may beisolated from a biological sample. The biological sample includes, butis not limited to, an oral plaque, saliva, gingival brush samples.Toll-like receptor reporter gene assays, such as TLR4-SEAP and/orTLR2-SEAP, may be used to detect and quantify bacterial toxins,including but not limited to endotoxins, in a biological sample.

A BODIPY TR cadaverine assay may be used to detect and quantifylipopolysaccharide in a biological sample. In addition, LAL (the Limulusamebocyte lysate assay) assay or endotoxin detection assay may be usedto detect and quantify the lipopolysaccharide in a biological sample.

The present invention may be directed toward a method for determiningthe potency of an oral biofilm comprising: a) providing a biofilmsample; b) providing reporter cells expressing one or more Toll likereceptors; c) exposing the cells to the biofilm sample; d) measuring theEC50 value of the biofilm activation of a Toll like receptor; e)quantification of the lipopolysaccharide. The biofilm may be an oralplaque, including but not limited to subgingival plaque, marginal orgumline plaque, supragingival plaque.

The present invention may also be directed toward a method fordetermining the potency of virulence in an oral sample comprising: a)providing an oral sample; b) providing reporter cells expressing one ormore Toll-like receptors; c) exposing the cells to the oral sample; d)measuring the EC50 value of the biofilm activation of a Toll likereceptor; e) quantification of the lipopolysaccharide. The oral samplemay include saliva, oral lavage or gingival crevicular fluid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing secretion of LPS during bacterial growth inculture.

FIG. 1B is a graph showing time course of bacterial growth in culturemedium.

FIG. 1C is a graph showing secretion of LPS during bacterial growth inculture.

FIG. 1D is a graph showing secretion of LPS during bacterial growth inculture at 24 hours. P. pallens and P. nigrescens released more LPS intothe medium than P gingivalis.

FIG. 1E is a graph showing the amount of LPS per bacterium duringbacterial growth in culture at 24 hours. P. pallens and P. nigrescenscontain more LPS than P gingivalis.

FIG. 1F is a picture showing the taking of gingival plaque samples usingPeriopaper.

FIG. 2A is a graph showing cell culture and virulence determination oflipopolysaccharide from different bacteria.

FIG. 2B is a graph showing activation of TLR4 reporter gene in culture.

FIG. 2C is a graph showing activation of TLR4 reporter gene in culture.

FIG. 3 is a graph showing stannous fluoride inhibition of TLR4 reportergene expression.

FIG. 4 is a graph showing stannous fluoride prevention of P. gingivalisLPS from activating TLR2 reporter gene expression.

FIG. 5 is a graph showing percentage reduction of P. gingivalis LPS onactivation of TLR-4 by stannous fluoride.

FIG. 6 is a graph showing EC50 determination of subgingival plaques thatactivate TLR4-SEAP signal transduction. Dental plaques from unhealthysites had a smaller EC50 than those from healthy sites, suggesting thedental plaques from unhealthy sites contain more virulence factors.

FIG. 7A is a graph showing TLR4 Evaluation of Human Subgingival Plaque(Healthy vs. Gingivitis).

FIG. 7B is a graph showing TLR4 Evaluation of Human oral lavage (Healthyvs. Gingivitis). and

FIG. 8A is a graph showing cell culture and virulence determination ofLTA from different bacteria.

FIG. 8B is a graph showing cell culture and virulence determination ofLPS from different bacteria.

FIG. 8C is a graph showing cell culture and virulence determination ofLPS from different bacteria.

FIG. 8D is a graph showing cell culture and virulence determination ofLPS from different bacteria.

FIG. 9 is an EC50 graph showing determination of subgingival plaquesthat activate TLR2-SEAP signal transduction.

FIG. 10A is a graph showing TLR2 Evaluation of Human Subgingival Plaque(Healthy vs. Gingivitis).

FIG. 10B is a graph showing TLR2 Evaluation of Human oral lavage(Healthy vs. Gingivitis).

FIG. 11 is a graph showing cell culture and virulence determination offlagellins from different bacteria.

FIG. 12A is a graph showing LPS Stimulated TLR Activity in HumanMonocytes THP-1.

FIG. 12B is a graph showing Evaluation of Human oral lavage (Healthy vs.Gingivitis) using the IRF-Luc reporter gene in THP-1 cells.

FIG. 12C is a graph showing Evaluation of Human Subgingival Plaque(Healthy vs. Gingivitis) in THP-1 cells using the NFkB reporter gene.

FIG. 13 is a graph showing detection of LTA of different bacteria inTHP1 dual reporter cells.

FIG. 14 is a graph showing clinical measurements.

FIG. 15 is graphs showing decreases in bacterial abundance in thesupragingival plaques during six weeks of regimen treatment.

FIG. 16 is a graph showing citrulline concentrations in Buccal-brushsamples over a six-week regimen treatment.

FIG. 17 is a graph showing protein bound ornithine in Buccal brushsamples during treatment.

FIG. 18 is a diagram showing enzymes in the ornithine, citrulline andarginine cycle.

FIG. 19 is a graph showing expression of ornithine, citrulline andarginine genes during a 6-week treatment.

FIG. 20 is a graph showing citrulline concentrations increased inBuccal-brush samples in experimental gingivitis.

FIG. 21 is graphs showing protein bound citrulline in Buccal brushsamples in experimentally induced gingivitis.

FIG. 22 is graphs showing concentrations of protein bound ornithine inBuccal-brush samples in experimentally induced gingivitis.

FIG. 23 is graphs showing concentrations of total ornithine inBuccal-brush samples in experimentally induced gingivitis.

FIG. 24 is graphs showing concentrations of protein bound arginine inBuccal-brush samples in experimentally induced gingivitis.

FIG. 25 is a graph showing concentrations of total arginine inBuccal-brush samples in experimentally induced gingivitis.

FIG. 26 is a graph showing citrulline inhibited cytokine productionstimulated by P. gingivalis LPS 1690 in THP-1 cells.

FIG. 27 is graphs showing bacterial LTA reduces fluorescent intensity ofBC in a dose dependent manner.

FIG. 28 is graphs showing LPS reduces the fluorescent intensity of BC ina dose-dependent manner.

FIG. 29 is graphs showing the supernatant of bacterial culture reducesthe fluorescent intensity of BC in a dose-dependent manner.

FIG. 30 is graphs showing bacteria reduce the fluorescent intensity ofBC in a dose-dependent manner.

FIG. 31 is graphs showing EC50 values.

DETAILED DESCRIPTION OF THE INVENTION

As disclosed herein, it was surprisingly discovered that one or moremethods could be used to detect and quantify the virulence insubgingival and supragingival plaques, thus distinguishing healthygingivae from an inflamed site suffering from the symptoms ofgingivitis. The state of health of the gingivae can be directly relatedto the level of bacterial toxins present, for example, endotoxins, andthus a reduction of these toxins, as discussed herein, in the oralcavity, as determined by screening on non-gingival engineered cells,would improve overall oral health. Additionally, the way an individualresponds to the virulence factors can be quantified utilizing theindividual's metabolic pathways, such as by quantifying products of theurea cycle.

Gingivitis is defined per the FDA monograph (12 CFR Part 356, Vol. 68,No. 103 (2003)) as “An inflammatory lesion of the gingiva that is mostfrequently caused by dental plaque. Gingivitis is characterized bytissue swelling and redness, loss of stippling (a normal state in whichthe surface of healthy gingiva is comprised of small lobes), glossysurface, and increased tissue temperature. The gingiva also may bleedupon gentle provocation, such as tooth brushing or may bleedspontaneously. Gingivitis is usually not painful.” Within the monograph,plaque is defined as being composed of multiple bacterial species. Thosespecies exert a constant inflammatory pressure on the host tissues.

When the inflammation progresses to the state of gingivitis, thereexists a need to quantify how severe the gingivitis is and how effectivetreatments from oral hygiene products are in reducing the inflammatoryresponse. The reduction in inflammatory response due to activation ofmembrane bound receptors across the gingival cells is termeddetoxification; and measuring the level of detoxification, which islacking in the art, is needed to educate consumers on the efficacy oftheir oral hygiene.

Pathogenesis of gingivitis involves both bacteria and host responses.The present invention discloses methodologies measuring the virulencefactors in the dental plaques in vitro, and also measuring effects ofvirulence factors on gingival tissues in vivo. These methodologies allowunderstanding of what virulence factor types are present in the dentalplaques, and how the host responds Importantly, the measuring ofvirulence factors provides a detailed assessment on the severity ofgingivitis in terms of virulence factors of the microbes in dentalplaques and the health status of the host. In addition, these methodshelp evaluate the effectiveness of a technology in preventing andtreating gingivitis.

The present invention includes a methodology comprising one or more ofthe following steps: (1) using the endotoxin detection assay kit orBODIPY-TR cadaverine method to quantify total LPS and LTA present in abiological sample and to detect technologies that inhibit the endotoxindetection assay; (2) using the Toll like receptor assays to determinepotency of purified virulence and oral dental plaques, and to measurethe efficacy of technologies that neutralize toxicity of virulencefactors; (3) using meta-sequencing to identify and quantitate bacteriain supragingival plaques, (4) measuring ornithine and citrulline ingingival swab samples to determine the healthy status of gingivaltissue, (5) measuring protein and mRNA levels to determine the levels ofhost responses, and determining citrulline activities in inhibitingLPS-induced production of proinflammatory cytokines.

The above-mentioned methods and chemistry may be applied in a strip formto the outer surface of the tooth and gumline. The strip may containcolor or fluorescence reagents to interact with the virulence factorspresent and thus allow for semi-quantative determination of thevirulence present. This would allow for rapid assessment of the level ofseverity of gingivitis and/or periodontal disease or to ascertain theeffectiveness of oral products. This execution of the described methodsallows for consumers to ascertain the state of their dental health athome or allows for professionals, such as dentists, to rapidly measurethe state of a patient's oral health.

The methods described above can be used to determine the cellular impactof organic and inorganic molecules, as long as there is an interactionbetween the molecule and the targeted receptor. Further, the methodscould be used to ascertain if a molecule would cause an irritation orinflammatory response. The receptor in question would be used in areporter system, as described herein, and the molecular impact of themolecule in question determined. The EC50 value of the molecule inquestion would be determined using the receptor associated with thebiological response, thus reducing or eliminating the need to do animaltesting.

All percentages and ratios used hereinafter are by weight of totalcomposition, unless otherwise indicated. All percentages, ratios, andlevels of ingredients referred to herein are based on the actual amountof the ingredient, and do not include solvents, fillers, or othermaterials with which the ingredient may be combined as a commerciallyavailable product, unless otherwise indicated.

All measurements referred to herein are made at 25° C. (i.e. roomtemperature) unless otherwise specified

As used herein, the word “include,” and its variants, are intended to benon-limiting, such that recitation of items in a list is not to theexclusion of other like items that may also be useful in the materials,compositions, devices, and methods of this invention.

As used herein, the word “or” when used as a connector of two or moreelements is meant to include the elements individually and incombination; for example X or Y, means X or Y or both.

By “personal care composition” is meant a product, which in the ordinarycourse of usage is applied to or contacted with a body surface toprovide a beneficial effect. Body surface includes skin, for exampledermal or mucosal; body surface also includes structures associated withthe body surface for example hair, teeth, or nails. Examples of personalcare compositions include a product applied to a human body forimproving appearance, cleansing, and odor control or general aesthetics.Non-limiting examples of personal care compositions include haircoloring compositions, oral care compositions, after shave gels andcreams, pre-shave preparations, shaving gels, creams, or foams,moisturizers and lotions, cough and cold compositions, leave-on skinlotions and creams, shampoos, conditioners, shower gels, bar soaps,toilet bars, antiperspirants, deodorants, depilatories, lipsticks,foundations, mascara, sunless tanners and sunscreen lotions.

By “oral care composition”, as used herein, is meant a product, which inthe ordinary course of usage, is not intentionally swallowed forpurposes of systemic administration of particular therapeutic agents,but is rather retained in the oral cavity for a time sufficient tocontact dental surfaces or oral tissues. Examples of oral carecompositions include dentifrice, mouth rinse, mousse, foam, mouth spray,lozenge, chewable tablet, chewing gum, tooth whitening strips, floss andfloss coatings, breath freshening dissolvable strips, or denture care oradhesive product. The oral care composition may also be incorporatedonto strips or films for direct application or attachment to oralsurfaces.

The term “dentifrice”, as used herein, includes tooth orsubgingival-paste, gel, or liquid formulations unless otherwisespecified. The dentifrice composition may be a single phase compositionor may be a combination of two or more separate dentifrice compositions.The dentifrice composition may be in any desired form, such as deepstriped, surface striped, multilayered, having a gel surrounding apaste, or any combination thereof. Each dentifrice composition in adentifrice comprising two or more separate dentifrice compositions maybe contained in a physically separated compartment of a dispenser anddispensed side-by-side.

The term “teeth”, as used herein, refers to natural teeth as well asartificial teeth or dental prosthesis.

Virulence factors are molecules produced by pathogenic microbes thatcontribute to the pathogenicity of the organism and enable them toinvade and proliferate in the host, and evade host immune surveillance.Virulence factors include, but are not limited to the following: Grampositive and Gram negative cell wall components, such aslipopolysaccharide and lipoteichoic acids, bacterial DNA, flagellin,peptidoglycan, adhesins, invasins, and antiphagocytic factors,hemolysins, bacterial metabolites, fimbriae, outer membrane vesicles,bacterial proteins or bacterial enzymes. A reduction in virulence or“detoxification”, based on reduced activation of Toll-like receptors,can be used to measure the effectiveness of various treatments.

The term “detoxification” or “detoxify” or “detox” as used herein,refers to the neutralization, reduction, or removal of microbialvirulence factors as measured by a reduction in the activation of areceptor known to be responsive with a virulence factor from anengineered cell, such as a reporter cell line. In certain embodiments adetermination of virulence is assigned based on the activation of one ormore of the Toll-like receptors.

The present invention includes obtaining a sample of oral matter. Theoral matter can include gum-line plaque, subgingival plaque,supragingival plaque, interstitial plaque, gingival crevicular fluid(GCF), gingival biopsy, saliva, or tongue swab. The oral matter may beobtained by any method known in the art, for example, subgingival plaquesample may be collected physically by scraping or by using paper points.The plaque may be collected off of the tooth beneath gums from thesulcus, the developed periodontal pocket, or at the gumline. Forexample, each paper point can be placed in the pocket between the toothand the gingiva for 10 seconds. After 10 seconds, a paper point can beremoved and placed into a pre-labeled 1.5 ml tube with 700 μlphosphate-buffered saline. The sampling procedure can be repeated withthree more paper points. After all four paper points are collected, the1.5 ml tube will be closed, vortexed for 30 seconds and placed on dryice until the samples are stored in a −80° C. freezer. Other methods ofcollection could include a mechanical device to help release the plaquefrom the tooth surface, such as a sonic descaler. The oral matter may beobtained both before and after treatment of an oral site from which theoral matter is obtained. An oral site from which the oral matter isobtained includes host tissues and bacterial matters. Further treatmentof an oral site may be more than once and may include multiple differenttreatments, for example a regimen, such as brushing teeth followed bymouthrinse. In addition to obtaining oral matter before and aftercompleted treatments, oral matter may be obtained between separatetreatments, for example between the brushing of teeth followed by theuse of mouth rinse.

The sample of oral matter is combined with a Toll-like receptor in areporter cell. Examples of Toll-like Receptors that can be used in thepresent invention include TLR 2, TLR 4, TLR 5, and TLR 9. Human TLR cDNA(TLR 1, 2, 3, 4, and 5) was first cloned in 1998 and their sequenceswere published (Rock F L, Hardiman G, Timans J C, Kastelein R A, Bazan JF. A family of human receptors structurally related to Drosophila Toll.Proc Natl Acad Sci USA. 1998 Jan. 20; 95:588-93). Rock et al. discoveredthat cytoplasmic domains of Drosophila Toll and human interleukin 1receptors shared high sequence homology, and hypothesized that bothmolecules trigger related signaling pathways tied to the nucleartranslocation of Rel-type transcription factors. They cloned a class ofputative human receptors with a protein architecture that is similar toDrosophila Toll in both intra- and extracellular segments. Theyhypothesized that the five human Toll-like receptors—named TLRs 1-5—areprobably the direct homologs of the fly molecule and, as such, couldconstitute an important and unrecognized component of innate immunity inhumans. Soon after the DNA sequences for TLR1 to 5 were published, otherTLR DNA sequences were also revealed. For example, TLR6 cDNA sequencewas reported in 1999 (Takeuchi O1, Kawai T, Sanjo H, Copeland N G,Gilbert D J, Jenkins N A, Takeda K, Akira S. A novel member of anexpanding toll-like receptor family. Gene. 1999 Apr. 29;231(1-2):59-65). cDNA sequences of human TLR7, TLR8 and TLR9 werereported in 2000 (Chuang TH1, Ulevitch R J. Cloning and characterizationof a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9.Eur Cytokine Netw. 2000 September; 11(3):372-8).

SEQ ID NO Sequence 1 Human Toll-Like Receptor 1 Amino Acid sequence 2Human Toll-Like Receptor 2 Amino Acid sequence 3 Human Toll-LikeReceptor 3 Amino Acid sequence 4 Human Toll-Like Receptor 4 Amino Acidsequence 5 Human Toll-Like Receptor 5 Amino Acid sequence 6 HumanToll-Like Receptor 6 Amino Acid sequence 7 Human Toll-Like Receptor 7Amino Acid sequence 8 Human Toll-Like Receptor 8 Amino Acid sequence 9Human Toll-Like Receptor 9 Amino Acid Sequence

A sequence listing that sets forth the amino acid sequences for SEQ IDNO: 1 to 9 herein is being filed concurrently with the presentapplication as an ASCII text file titled“13837M_AA_Sequence_Listing_ST25.” The ASCII text file was created on 29Mar. 2016 and is 68 Kbytes in size. In accordance with MPEP §605.08 and37 CFR §1.52(e), the subject matter in the ASCII text file isincorporated herein by reference.

TLR proteins can form heterodimers or homodimers. There are 10 TLR genesidentified in humans. Their gene products form homodimers orheterodimers in cell membranes. For example, TLR1 can form heterodimerswith TLR. Similarly, TLR6 also can assemble a heterodimer with TLR2. Onother hand, TLR4 forms a homodimer. Toll-like receptors (TLRs) are aclass of proteins that play a key role in the innate immune system aswell as the digestive system. They are membrane-spanning, non-catalyticreceptors usually expressed in cells such as macrophages, dendritecells, and gingival epithelial cells that recognize structurallyconserved molecules derived from microbes. Once these microbes havebreached physical barriers such as the skin or intestinal tract mucosaor oral epithelial cells they are recognized by TLRs, which activateimmune cell responses. Toll-like receptors were targeted because theyare the primary sensors of virulence factors produced by the microbes inthe dental plaques. TLR 1, 2, 4, 5 and 6 are expressed in the cellplasma membranes, providing easy access for the virulence factors to besensed by the host receptors. TLR 3, 7, 8, and 9 are located on themembranes of endosomes; and as virulence factors are phagocytosed intothe cells, they also gain access to TLR 3, 7, 8 and 9

A reporter cell refers to a eukaryotic cell, such as, but not limitedto, HEK 293T, human monocyte (THP1), Chinese hamster ovary (CHO) cell,murine cells, or monkey kidney epithelial (Vero) cells engineered toexpress a predetermined number of TLR receptors, for example a singleTLR receptor; which is in contrast to gingival cells that expressmultiple functional TLR receptors. Thus, one type of engineered reportercells respond to only one type of virulence factors in the dentalplaques. In contrast, gingival cells express several types of functionalTLRs, and can't be used to identify a single type of virulence factor indental plaques. The output from gingival cells is the sum of variousvirulence factors in the dental plaques. HEK 293T cells can be used asreporter cells, as they are easy to maintain and have similar geneexpression profiles to oral epithelial cells, making them a closer matchto the gene expression of gingival cells, such that the results willmirror in vivo results. In contrast to naturally occurring gingivalcells the reporter cells of the present invention are easy to maintainin the lab, and stable in phenotypes. Further, the reporter cells makedetection of virulence factors simpler, are more reproducible, increaseaccuracy, provide higher throughput are more specific and morequantifiable.

Individual TLR receptor genes are stably transfected to HEK 293 cells asdescribed by Invivogen (/PDF/HEK_Blue_htlr4_TDS.pdf). HEK-Blue™ hTLR4Cells are designed for studying the stimulation of human TLR4 (hTLR4) bymonitoring the activation of NF-kB. HEK-Blue™ hTLR4 Cells were obtainedby co-transfection of the hTLR4 gene, the MD-2/CD14 co-receptor genesand a secreted embryonic alkaline phosphatase (SEAP) reporter gene intoHEK293 cells. The SEAP reporter gene is placed under the control of anIL-12 p40 minimal promoter fused to five NF-kB and AP-1-binding sites(Supplement 1. HEK-Blue™ hTLR4 Cells SEAP Reporter 293 cells expressingthe human TLR4 gene, Catalog # hkb-htlr4, Version #15C04-MM(/PDF/HEK_Blue_htlr4_TDS.pdf). As compared to measuring an immuneresponse the reporter genes of the present invention allow rapid,specific and reproducible measurements of virulence factors.

The level of receptor activation can be determined by any method knownin the art for the type of reporter gene used. For example if anNFkB-SEAP reporter gene is used, one could measure the production ofSEAP in the culture medium. The reporter cells can be treated withvirulence factors, or dental plaque matters collected before or aftertreatments. Expression of the reporter gene will be stimulated and SEAPsecreted into the medium when stimulated by virulence factors. The levelof reporter gene product SEAP can be readily measured with commercialkits, and will be proportional to the amount of particular types ofvirulence factors. Similarly, if an NFkB-luciferase,NFkB-beta-lactamase, or other reporter genes are used, available kitscan be used to measure the reporter gene products.

The potency can then be determined based on parameters such as, EC50 andfold of stimulation. EC50 provides a measurement on the amount ofvirulence factors needed to mount an inflammatory response, and the foldof stimulation is indicative of the severity of inflammatory responsesthe virulence factors cause. The EC50 is used to determine potency;wherein “potency” as defined by the Merck Manual, refers to theconcentration (EC50) of a chemistry required to produce 50% of thechemistry's maximal effect as depicted by a graded dose-response curve.EC50 equals Kd (Dissociation constant, which is a measure of 50% of thesubstance in question bound to the receptor) when there is a linearrelationship between occupancy and response. Often, signal amplificationoccurs between receptor occupancy and response, which results in theEC50 for response being much less (ie, positioned to the left on theabscissa of the log dose-response curve) than Kd for receptor occupancy.Potency depends on both the affinity of a compound for its receptor, andthe efficiency with which a compound-receptor interaction is coupled toresponse. The dose of a compound required to produce an effect isinversely related to potency. In general, low potency is important onlyif it results in a need to administer a compound in large doses that areimpractical. Quantal dose-response curves provide information on thepotency of a compound that is different from the information derivedfrom graded dose-response curves. In a quantal dose-responserelationship, the EC50 is the dose at which 50% of individuals exhibitthe specified quantal effect.

In the present invention, the activities of endotoxins orlipopolysaccharides can be measured using an endotoxin detection assay,or LAL assay. The Limulus Amebocyte Lysate (LAL) test has been used todetect LPS. LAL is derived from the blood cells, or amebocytes, of thehorseshoe crab, Limulus polyphemus. At present, some main endotoxindetection agents are derived from recombinant proteins. Thereinafter,endotoxin detection assay and LAL assay are used interchangeably.

-   A. A method of determining bacterial virulence in an oral cavity    comprising:    -   a. providing a reporter cell expressing at least one toll-like        receptor;    -   b. providing a sample of oral matter;    -   c. combining the sample of oral matter and the reporter cell;    -   d. measuring the toll-like receptor activation.-   B. The method of paragraph A, wherein the toll-like receptor is at    least one of TLR1 to TLR9.-   C. The method according to paragraph A or B, wherein the reporter    cell expresses at least of one TLR2 or TLR4.-   D. The method according to any one of paragraphs A to C, wherein the    toll-like receptor activates a reporter gene, preferably wherein the    reported gene is a secreted embryonic alkaline phosphatase (SEAP)    reporter gene.-   E. The method according to any one of paragraphs A to D, wherein the    sample of oral matter is at least one of gum-line plaque,    subgingival plaque, supragingival plaque, interstitial plaque,    gingival crevicular fluid (GCF), gingival biopsy, saliva, or tongue    swab, preferably wherein the sample of oral matter comprises    virulence factor that is at least one of Gram positive cell wall    components, Gram negative cell wall components, bacterial DNA,    flagellin, peptidoglycan, bacterial metabolites, fimbriae; outer    membrane vesicles bacterial proteins, or bacterial enzymes.-   F. The method of paragraph E, wherein the sample of oral matter    comprises virulence factor that is at least one of    lipopolysaccharide or lipoteichoic acid.-   G. The method according to any of paragraphs A to F comprising the    additional steps of:    -   e. providing another sample of oral matter from an individual        who used an oral care composition prior to providing another        sample of oral matter;    -   f. combining the another sample of oral matter and the reporter        cell;    -   g. measuring the toll-like receptor response;    -   h. comparing the toll-like receptor response from the sample of        oral matter and another sample of oral matter.-   H. The method of paragraph G, where the sample of oral matter and    the another sample of oral matter are from the same individual.-   I. A method for determining the virulence of lipopolysaccharide    comprising:    -   a. providing a lipopolysaccharide;    -   b. providing a reporter cell expressing at least one Toll-like        receptor;    -   c. combining the reporter cell with the lipopolysaccharide;    -   d. measuring the toll-like receptor activation;    -   e. quantifying the lipopolysaccharide.-   J. The method of paragraph I, wherein the Toll-like receptor    activation is determined by measuring at least one of EC50 or fold    of stimulation.-   K. The method according to paragraph I or J, wherein the Toll-like    receptor is at least one of TLR1 to TLR9.-   L. The method according to any of paragraphs I to K, wherein the    reporter cell expresses at least one of TLR2 or TLR4.-   M. The method according to any of paragraphs I to L, wherein the    Toll-like receptor activates a reporter gene.-   N. The method according to paragraph M, wherein the reported gene is    a secreted embryonic alkaline phosphatase (SEAP) reporter gene.-   O. The method according to any of paragraphs I to N, wherein the    lipopolysaccharide is quantified using at least one of limulus    amebocyte lysate assay or BODIPY TR cadaverine assay.

EXAMPLES Example 1 Determination of Secreted Lipopolysaccharide byBacteria

Growth of bacteria: A 1 ml aliquot of a 24 hour culture of E. coli ATCC8739 was used to inoculate 100 ml of Luria-Bertani (LB) broth in a 250ml baffled flask. This culture was then incubated at 37° C. withagitation (220 rpm) and sampled at 30 minute intervals. Samples wereassessed for turbidity (OD₆₀₀) in a SpectraMax platereader M3 (MolecularDevices, Sunnydale, Calif.), which is one method of monitoring thegrowth and physiological state of microorganisms. The sample turbiditywas then recorded and the samples were centrifuged at 5000 RPM for 10min at room temperature. The supernatant, thereinafter referred to as“supernatant of bacterial culture”, was subsequently analyzed for LPScontent using the procedure as described below.

Twenty ml aliquots of MTGE broth (Anaerobe Systems, Morgan Hill, Calif.)were inoculated with P. gingivalis ATCC 33277, P. pallens ATCC 700821,or P. nigrescens ATCC 25261. These cultures were incubated overnight ina Whitely A45 Anaerobic Workstation (Don Whitley Scientific, Frederick,Md.) at 37° C. with an 85:10:5 N₂:CO₂:H₂ gas ratio. One ml aliquots ofthese starter cultures were then used to inoculate 99 ml ofmembrane-Tryptone Glucose Extract (m-TGE) broth in a 250 ml baffledflask. These cultures were then incubated under agitation (200 rpm) aspreviously described and sampled at regular intervals. Samples wereassessed for turbidity (OD₆₀₀) in a Tecan Infinite m200 Pro (TecanTrading AG, Switzerland) and then centrifuged at 16,100×g for 10 min atroom temperature. Supernatants were decanted and passed through a 0.22μM filter prior to analysis for LPS content.

In the experiment, only OD600 was measured. For the sake of consistencyin following experiments, we converted OD600 readings into bacterialnumbers, even though the relationship between OD600 readings andbacterial numbers is varied for each bacterium. The number of bacteriawas estimated based on spectrophotometer readings at OD₆₀₀ (OD₆₀₀ of1.0=8×10⁸ cells/ml).

The Limulus Amebocyte Lysate Assay (LAL) is an assay to determine thetotal amount of lipopolysaccharide (LPS) in the sample tested (PierceLAL Chromogenic Endotoxin Quantitation Kit, ThermoFischer Scientific,Waltham, Mass.). The assay was performed following manufacturer'sinstruction. Ninety-six-well microplates were first equilibrated in aheating block for 10 min at 37° C. Fifty μl each of standard or samplewas dispensed into the microplate wells and incubated with plate coveredfor 5 min at 37° C. Then 50 μl LAL was added to each well. Plates wereshaken gently and incubated for 10 min at 37° C. 100 μl of chromogenicsubstrate was added and incubated for 6 min at 37° C. Finally, 50 μlStop Reagent was added and the absorbance was measured at 405-410 nm onSpectramax M3 platereader (Molecular Device, Sunnyvale, Calif.).

FIGS. 1A, 1C, and 1D show the ability of microbes to shed LPS as part oftheir normal growth cycle. This data shows the need to deliver chemistryto the subgingival plaque to effectively mitigate the LPS, since toothbrushing generally does not remove the subgingival plaque.

The LPS, as measured by the LAL kit reported in endotoxin unit per ml(EU/ml), was shed by the bacteria (E. coli K12) as depicted in FIG. 1A.The growth media began to be depleted of complex sugars around 120minutes, as reflected in the bacterial growth curve in FIG. 1B, wherethe LPS shedding started to decline. This data gave a reason to believethat a mature biofilm/plaque could supply a constant level of LPS to thehost cells, if food sources were present. The LPS would then have theability to induce an inflammatory response from the host cells.

Importantly, LPS are secreted into the supernatant of bacterial culture(FIG. 1D). LPS also exists in bacterial walls (FIG. 1E). Again, thisdata further enforce the need to deliver chemistry to the subgingivalplaque to effectively mitigate the LPS, since tooth brushing generallydoes not remove the subgingival plaque.

Example 2 LAL Assay to Quantify Virulence Factors in Dental Plaques

Seven panelists, with at least three bleeding sites, took part in thetesting. A licensed dental hygienist collected subgingival plaquesamples. Samples were taken at the tooth/gum interface (buccal surfacesonly) using care to avoid contact with the oral soft tissues. Sixsubgingival plaque sites were sampled from each panelist (3 healthy and3 unhealthy sites). Unhealthy teeth had bleeding sites with pocketsgreater than 3 mm and healthy sites had no bleeding with pocket depthless than 2 mm Prior to sampling, panelists were instructed to abstainfor 12 hours from oral hygiene and refrain from eating, chewing gum,drinking (except small sips of water). Next, panelists had theirmarginal plaque collected with a curette at the sampling sites. Then,from the same site, subgingival plaque samples were collected with 3consecutive paper points as shown in FIG. 1F. The sampling sites wereisolated with cotton rolls and gently air-dried. Paper points (PROFLOWincorporated, Amityville, N.Y.) were gently placed for 10 seconds intothe pocket until a minimum of resistance was felt. After 10 seconds,paper points were removed and placed into pre-labeled 1.5 ml tubes. Thesame sampling procedure was repeated with 2 more paper points (paperpoints go into separate tubes). The first, second and third sample paperpoints from a healthy site of all panelists were pooled separately intothree tubes, labeled as paper point 1, 2 and 3, respectively. Similarlythe unhealthy site samples were also pooled.

TABLE 1 showed that unhealthy dental plaques contained more endotoxinsthan the healthy dental plaques. One ml PBS was added to each pooledsample in the 1.5 ml tube. Bacteria were lysed in a MolBio Fast Prepbead beater (MP Biomedicals, Santa Ana, Calif.). Samples werecentrifuged for 10 min at 10,000 RPM at 4° C., supernatants werecollected and analyzed with LAL assay kits following manufacturer'sinstruction as described in EXAMPLE 1.

TABLE 1 Protein concentrations and endotoxin levels in the pooled dentalplaque samples. Endotoxin Dental plaque (endotoxin unit) Healthypaperpoint 1 sub plaque 1284 Healthy paperpoint 2 sub plaque 476 Healthypaperpoint 3 sub plaque 361 Healthy Marginal Plaque 23180 Unhealthypaperpoint 1 sub plaque 3371 Unhealthy paperpoint 2 sub plaque 1732Unhealthy paperpoint 3 sub plaque 1644 Unhealthy Marginal Plaque 80277

It was expected that the marginal plaques in unhealthy sites had moreendotoxins than those in the healthy sites (TABLE1) within the samesubjects. Three samples were taken from subgingival pockets with threepaper points sequentially, named paper point 1, 2 and 3. Again, thesubgingival plaques taken by the paper point 1 had more endotoxins inthe unhealthy sites than in the healthy sites (TABLE 1). The same istrue for the samples taken by paper point 2 and 3 Importantly, dentalplaques in the unhealthy subgingival pockets possessed more endotoxinsthan plaques from healthy pockets. This may explain why unhealthygingiva are prone to bleeding upon probing.

Example 3 Technology Development Using the LAL Assay Kit

The LAL assay, as described in EXAMPLE 1, was modified for developmentof technology which inhibits LPS from activating a proenzyme in the LALassay. The Thermo Scientific Pierce LAL Chromogenic EndotoxinQuantitation Kit is a quantitative endpoint assay for the detection ofLPS, which catalyzes the activation of a proenzyme in the modifiedLimulus Amebocyte Lysate (LAL). The activated proenzyme then splitsp-Nitroaniline (pNA) from the colorless substrate,Ac-Ile-Glu-Ala-Arg-pNA. The product pNA is photometrically measured at405-410 nm. If SnF2 binds to LPS, the latter can't react with theproenzyme in the LAL kit. Consequently, the proenzyme is not activated,and the colorless substrate Ac-Ile-Glu-Ala-Arg-pNA will not split and nocolor product is produced. P. gingivalis LPS 1690 (1 ng/ml), or E. coliLPS (1 ng/ml), and stannous fluoride and other materials (50 and 500μM), as listed in TABLE 2, were dissolved in endotoxin-free water. Then50 μl LAL was added to each well. Plates were shaken gently andincubated for 10 min at 37° C. 100 μl of chromogenic substrate was addedand incubated for 6 min at 37° C. Finally, 50 μl Stop Reagent was addedand the absorbance was measured at 405-410 nm on Spectramax M3 platereader (Molecular Device, Sunnyvale, Calif.).

As shown in TABLE 2, SnF2 and some other compounds inhibited LPSactivities in LAL assays

TABLE 2 Inhibition of LPS activities on LAL Assays Inhibition of LALactivity % P. gingivalis LPS E. coli LPS 1690 1 ng/ml 1 ng/ml Samples500 uM 50 uM 500 uM 50 uM Tin (II) fluoride 60 49 92 87 stannouschloride 48 21 89 65 Cetylpyridinium chloride 103 77 103 46 monohydrateChlorhexidine 102 38 97 57 zinc citrate, dihydrate 104 57 104 82 zinclactate 58 0 66 0 potassium oxalate 8 0 1 6 Triclosan (irgasan) 0 0 10 01-Hydroxypyridine-2-thone 0 0 2 6 zinc salt sodium fluoride 0 0 4 5Carboxymethyl cellulose 0 0 2 0 sodium

Example 4 Binding of BODIPY-TR-Cadaverine to LPS and Displacement of LPSBinding by Stannous Fluoride

In addition to LAL quantification of LPS, the BODIPY method can beutilized to assess the level of LPS. Detoxifying technologies are ableto target and neutralize bacterial virulence factors, such as LPS andLTA. To develop such LPS and LTA sequestration technologies, a highthroughput screening was employed to identify molecules that disrupt theactivation of a Toll-like receptor by LPS and LTA, and other virulencefactors. The high throughput screen utilizes the fluorescent dyeBODIPY-TR-cadaverine5-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino)pentylaminehydrochloride (BC fluorescent dye), purchased from Life Technologies(Carlsbad, Calif.), as described previously by Wood, Miller and David(2004) (Comb Chem High Throughput Screen 2004 May; 7(3):239-49.Anti-endotoxin agents. 1. Development of a fluorescent probedisplacement method optimized for the rapid identification oflipopolysaccharide-binding agents.). The experiment was carried out at22° C. E. coli LPS (15 μg/ml) was mixed in a well of a 96-well solidblack flat bottom plate (Corning Incorporated, Corning, N.Y.) with SNF2and cetylpyridinium chloride in 30 μl of 50 mM Tris buffer at pH 7.4 for10 mM, and then 20 μl of 60 μM BC fluorescent dye was added right beforefluorescence measurement at 22° C., or room temperature. Fluorescencemeasurements were performed in a SpectraMax M3 automated 96 well platereader (Molecular Device, Sunnyvale, Calif.). The excitation wavelengthwas 580 nM and the fluorescence emission was measured at 620 nM.

As shown in TABLE 3, leading anti-gingivitis technologies, such asstannous fluoride, displaced LPS from BODIPY TR cadaverine.

TABLE 3 Displacement of LPS from binding to BODIPY TR cadaverineDisplacement % Concentrations mM Stannous fluoride Cetylpyridiniumchloride 0.16 2.45 9.98 0.31 2.79 31.11 0.63 12.69 68.05 1.25 49.4491.12

Example 5 Cell Culture and Virulence Determination of Lipopolysaccharidefrom Different Bacteria

Reporter gene cell lines, human HEK 293T cells, were purchased fromInvivogen of San Diego, Calif. The HEK 293T cells were stablytransfected with at least two exogenous genes, a TLR4 structural gene,and a SEAP reporter gene, which is under the control of NFkBtranscriptional factors. The cell line is named here as TLR4-SEAP. Thereporter gene encodes a secreted enzyme, called embryonic alkalinephosphatase or SEAP. The SEAP reporter is placed under the control ofthe interferon-β minimal promoter fused to five NFkB and AP-1-bindingsites. Furthermore, the TLR4-SEAP cell line also contains a CD14co-receptor gene, which is needed to transfer LPS to TLR4 receptors. Therecombinant TLR binds its ligand, or distinct pathogen-associatedmolecule, initiates a chain of responses, leading to recruitment of NFkBand AP1 transcription factors to the reporter gene promoter, whichinduce expression of SEAP.

Cell culture and treatment: 500,000 gene reporter cells were grown andmaintained in 15 ml growth medium, comprised of DMEM medium supplementedwith 10% fetal calf serum in T75 flasks for three days at 37° C., 5%CO₂, and 95% humidity. For treatment, wells of a 96-well plate wereseeded with 10,000 cells/well in 100 μL, of growth medium. The cellswere incubated for 72 hours at 37° C., 5% CO₂, and 95% humidity untilday 4. On day 4, medium was changed to assay medium (90 μl), which isthe DMEM medium without fetal calf serum. LPS, bacteria and the culturemedium of bacterial growth, as described in EXAMPLE 1, were firstresolved or mixed with the assay medium. 10 μl of the bacteria, LPS andculture medium of bacterial growth were added to the TLR4-SEAP cells.Samples were taken 24 hours later, following addition of LPS, bacteria,and culture medium. Expression of the reporter gene (SEAP) wasquantified with a commercially available kit (SEAP Reporter Gene Assayof Cayman Chemical Co., Ann Arbor, Mich.).

EC50 was calculated using GraphPad Prism software (GraphPad Software, LaJolla, Calif.). Samples with lower EC50 are more potent in activatingthe TLR4 reporter gene than those with higher EC50. As shown in FIG. 2A,LPS from E. coli has lower EC50 than P. gingivalis, thus, was far morepotent than P. gingivalis (Pg). Salmonella Minnesota LPS is not aspotent as that of E. coli, but is far more potent than those of P.gingivalis LPS 1690 and 1435. Each species of bacteria produces multipleforms of LPS. Each form of LPS from the same species of bacteria hasdifferent potency in stimulating TLR4-downstream signaling pathways. Forexample, Pg 1690 LPS is more potent than Pg1435/50. LPS is a componentin bacterial cell walls. Likely, E. coli cell wall is more virulent ininducing production of proinflammatory cytokines in host cells than P.gingivalis when they are in direct contact with host blood cells. P.gingivalis had far higher EC50 than P. pallens and P. nigrescens asshown in FIG. 2B in stimulating TLR4 reporter gene expression,suggesting that P. pallens and P. nigrescens are more likely to promoteproduction of proinflammatory cytokines in host cells than P.gingivalis.

Bacteria release LPS into the supernatant of bacterial culture. As shownin FIG. 2C, the supernatant of P. pallens has an EC50 that is similar tothat of P. nigrescens, but far lower than that of P. gingivalis, instimulating expression of TLR4 reporter gene. Again, those results implythat the products of P. pallens and P. nigrescens are more likely topromote production of proinflammatory cytokines in host cells than thoseof P. gingivalis.

Example 6 Development of Technology for Inhibition of LPS Effects onStimulating Expression of NFkB-Regulated Reporter Genes

Stannous fluoride is a leading anti-gingivitis technology in P&Gtoothpaste products. Tests were conducted to understand whether stannousfluoride could reduce LPS's ability to trigger proinflammatory responsesin host cells. TLR4-SEAP reporter cells were prepared using the sameconditions as described in EXAMPLE 5 in the presence or absence of LPS.Production of SEAP was quantified also as described in EXAMPLE 5.

FIG. 3 shows the effect of stannous at various concentrations from 62.5uM to 1,000 uM on 100 ng/ml E. coli LPS, as reported by activation ofTLR-4. At stannous concentrations of 500 uM or higher, the level of E.coli induction of TLR-4 was decreased.

FIG. 4 shows the effects of stannous at various concentrations from 62.5uM to 1,000 uM on P. gingivalis LPS, as reported by activation of TLR-2.At stannous concentrations of 1000 uM, the level of P. gingivalisinduction of TLR-2 was decreased.

The data in FIG. 5 shows reduction of LPS activity by the stannous ion,from a stannous fluoride salt. The data showed that stannous fluoride,at 1.6 mM and 3.2 mM, reduce about 50% of P. gingivalis LPS (500 ng/ml)activation on the TLR4 reporter system (One asterisk means P<0.05, twoasterisks mean P<0.01).

Example 7 Cell Culture and Virulence Determination of EC50 in Activationof TLR4-SEAP Signal Transduction by Clinical Samples

The method described in EXAMPLE 5 is effective at determining thepotency of LPS from different bacteria. The same method was used todetermine the EC50 of clinical samples, as described in EXAMPLE 2. Asshown in FIG. 6, dental plaques from unhealthy sites had a smaller EC50than those from healthy sites, suggesting the dental plaques fromunhealthy sites contain more virulence factors.

The same method described in EXAMPLE 5 was used to examine the clinicalsamples in another study. A clinical study was conducted to evaluatesample collection methods and measurement procedures. It was acontrolled, examiner-blind study. Forty panelists met the inclusioncriteria, wherein in order to be included in the study, each panelistmust:

-   -   Provide written informed consent to participate in the study;    -   Be 18 years of age or older;    -   Agree not to participate in any other oral/dental product        studies during the course of this study;    -   Agree to delay any elective dentistry (including dental        prophylaxis) until the study has been completed;    -   Agree to refrain from any form of non-specified oral hygiene        during the treatment periods, including but not limited to the        use of products such as floss or whitening products;    -   Agree to return for all scheduled visits and follow study        procedures;    -   Must have at least 16 natural teeth;    -   Be in good general health, as determined by the        Investigator/designee based on a review of the health        history/update for participation in the study.

For Unhealthy Group (high bleeder group):

-   -   Have at least 20 bleeding sites (sites with a score of 1 or 2 on        the GBI index); Have minimum 3 sampling sites with bleeding and        pocket depth >3 mm but not deeper than 4 mm;    -   Have minimum 3 sampling sites without bleeding and with pocket        depth <2 mm

For Healthy Group (low bleeder group):

-   -   Have maximum 3 bleeding sites (sites with a score of 1 or 2 on        the GBI index);    -   No pockets deeper than 2 mm. Twenty (20) panelists were        qualified as healthy—with up to 3 bleeding sites and with all        pockets less than or equal to 2 mm deep and twenty (20)        panelists were qualified as unhealthy—with greater than 20        bleeding sites with at least 3 pockets greater than or equal to        3 mm but not deeper than 4 mm with bleeding, and at least 3        pockets less than or equal to 2 mm deep with no bleeding for        sampling. All panelists had up to 6 sites identified as        “sampling sites.” The “sampling sites” had supragingival and        subgingival plaque collected at Baseline, Week 2 and Week 4.        Subgingival plaque samples were taken from a gingival sulcus        from the pre-identified sites. Prior to sample collection, the        site had supragingival plaque removed with a curette. The site        was dried and subgingival plaque samples were collected with        another dental curette (e.g., Gracey 13/14, 15/16, 11/12, ⅞, ½.)        Each Gracey curette is designed to adapt to a specific area or        tooth surface. For example, Gracey 13/14 is designed to adapt to        the distal surfaces of posterior teeth. Samples from each site        were placed in a pre-labeled 2.0 ml sterile tube containing 300        μl of DPBS buffer with about 50 of sterile 1 mm glass beads.        Samples were stored at 4° C. The subgingival samples were stored        at −80° C. until analyzed. The samples were thawed at room        temperature and dispersed in a TissueLyser II (Qiagen, Valencia,        Calif., USA) at 30 shakes per second for 3 min Protein        concentrations of the dispersed subgingival samples were        measured using a Pierce microBCA Protein kit (ThermoFisher        Scientific, Grand Island, N.Y., USA) following the        manufacturer's instruction.

Oral lavage samples were collected at wake up (one per panelist) byrinsing with 4 ml of water for 30 seconds and then expectorating thecontents of the mouth into a centrifuge tube. These samples were frozenat home until they were brought into the site in a cold pack. Eachpanelist collected up to 15 samples throughout the study. Saliva sampleswere frozen at −70° C. from submission.

All panelists were given investigational products: Crest® Pro-HealthClinical Gum Protection Toothpaste (0.454% stannous fluoride) andOral-B® Indicator Soft Manual Toothbrush. Panelists continued theirregular oral hygiene routine, and did not use any new products startingfrom the baseline to the end of four week treatment study. During thefour week treatment period, panelists brushed their teeth twice daily,morning and evening, in their customary manner using the assigneddentifrice and soft manual toothbrush.

The subgingival plaques from the above clinical study were applied tothe TLR4 reporter cells in a procedure as described in EXAMPLE 5. FIG.7A shows the results of a four-week study of 40 panelists going frombaseline out over four weeks of treatment with Crest ProHealth Clinicaltoothpaste. The subgingival plaque samples in bleeding sites on the highbleeders group stimulated high expression of TLR4 reporter gene. Morevirulence in a sample elicits higher RLU (relative luminescent units)readings in the TLR4 reporter gene assay. As shown in FIG. 7A, thebaseline samples of the high bleeders group had higher RLU than those ofthe low bleeders on both the bleeding and non-bleeding sites. Aftertreatment with Crest® Pro-Health Clinical Gum Protection Toothpaste(0.454% stannous fluoride) and Oral-B® Indicator Soft Manual Toothbrushfor four weeks, the virulence was reduced at week 4 in both high andlower bleeders groups at both bleeding and non-bleeding sites.

The oral lavage samples were applied to the TLR4 reporter cells in aprocedure as described in EXAMPLE 5. As shown in FIG. 7B, oral lavage(Healthy vs. Gingivitis) samples were evaluated in the TLR4-SEAPreporter assay. The baseline samples of the high bleeders group hadhigher RLU than those of the low bleeders. After treatment with Crest®Pro-Health Clinical Gum Protection Toothpaste (0.454% stannous fluoride)and Oral-B® Indicator Soft Manual Toothbrush for four weeks, thevirulence was reduced at week 4 in the high bleeder group.

Example 8 Cell Culture and Virulence Determination of Lipoteichoic Acidsfrom Different Bacteria

The reporter gene cell lines, human HEK 293T cells, were purchased fromInvivogen of San Diego, Calif. The HEK 293T cells were stablytransfected with at least two exogenous genes, a TLR2 structural gene,and SEAP reporter gene which is under the control of NFkBtranscriptional factors. The cell line is named here as TLR2-SEAP. Thereporter gene encodes a secreted enzyme, called embryonic alkalinephosphatase or SEAP. The SEAP reporter is placed under the control ofthe interferon-0 minimal promoter fused to five NFkB and AP-1-bindingsites. Furthermore, a CD14 co-receptor gene was transfected into thereporter gene cells expressing TLR2, as CD14 has been identified as aco-receptor for TLR2 ligands to enhance the TLR response. The CD14co-receptor is needed to transfer LTA to TLR2 receptors. The recombinantTLR2 binds its ligand, or distinct pathogen-associated molecule,initiates a chain of responses, leading to recruitment of NFkB and AP1transcription factors to the reporter gene promoter, which induceexpression of SEAP.

Cell culture and treatment: 500,000 gene reporter cells were grown andmaintained in 15 ml growth medium, comprising DMEM medium supplementedwith 10% fetal calf serum in T75 flasks for three days at 37° C., 5%CO₂, and 95% humidity. For treatment with LTA, wells of a 96-well platewere seeded with 10,000 cells/well in 100 μL, of growth medium. Thecells were incubated for 72 hours at 37° C., 5% CO₂, and 95% humidityuntil day 4. On day 4, medium (100 μL) was changed to DMEM mediumwithout fetal calf serum. LTA, LPS and bacterial cells, as described inEXAMPLE 7, were added. Samples were taken 24 hours later, followingaddition of samples. Expression of the reporter gene (SEAP) wasquantified with a commercially available kit (SEAP Reporter Gene Assayof Cayman Chemical Co., Ann Arbor, Mich.).

As shown in FIGS. 8A, 8B, 8C and 8D, LTA, LPS, bacteria and thesupernatant of bacterial culture could bind to TLR2 and activate TLR2downstream signaling pathways in a dose-dependent manner. As shown inFIG. 8A, B. subtilis (BS) LTA is more potent than that of Enterococccushirae. As shown in FIG. 8B, P. gingivalis LPS also activated expressionof the TLR2 reporter gene. For example, Pg1690, as shown in FIG. 8B,activated TLR2-SEAP signal transduction, and stimulated SEAP production.But as shown in FIG. 8B, E. coli LPS did not activate the TLR2-SEAPreporter cells. It should also be noted that P. pallens, P. nigrescensand P. gingivalis have similar EC50 in stimulating expression of TLR2reporter gene (FIG. 8C). However, the released TLR2 ligands from thethree different bacteria have very different EC50 on activation of TLR2reporter gene (FIG. 8D).

Example 9 Cell Culture and Virulence Determination of EC50 in Activationof TLR2-SEAP Signal Transduction by Clinical Samples

The method described in EXAMPLE 8 is effective in determining the EC50of LTA and other TLR2 ligands from different bacteria. The same methodwas used to determine the EC50 of clinical samples, as described inEXAMPLE 2. As shown in FIG. 9, dental plaques from unhealthy (bleeding)sites had smaller EC50 than those from healthy (non-bleeding) sites,suggesting the dental plaques from unhealthy sites contain morevirulence factors.

Clinical samples as described for FIG. 7A of EXAMPLE 7 were examinedusing the TLR2-SEAP reporter gene assay. The results are shown in FIG.10A. The subgingival samples in unhealthy (bleeding) sites from theunhealthy group (high bleeders) had more virulence factors than othersites. The baseline samples of the high bleeders group had higher RLUthan those of the low bleeders on both the bleeding and non-bleedingsites. After treatment with Crest® Pro-Health Clinical Gum ProtectionToothpaste (0.454% stannous fluoride) and Oral-B® Indicator Soft ManualToothbrush for four weeks, the virulence was reduced at week 4 in bothhigh and low bleeders groups at both bleeding sites.

The clinical samples as described for FIG. 7B of EXAMPLE 7 were examinedusing the TLR2-SEAP reporter gene assay. As shown in FIG. 10B, orallavage (Healthy vs. Gingivitis) was evaluated. After treatment withCrest® Pro-Health Clinical Gum Protection Toothpaste (0.454% stannousfluoride) and Oral-B® Indicator Soft Manual Toothbrush for four weeks,the virulence was reduced at week 4 in the high bleeder group.

Example 10 Cell Culture and Virulence Determination of Flagellins fromDifferent Bacteria

The reporter gene cell lines, human HEK 293T cells, were purchased fromInvivogen of San Diego Calif. The HEK 293T cells were stably transfectedwith two exogenous genes, a TLR5 structural gene, and SEAP reporter genewhich is under the control of NFkB transcriptional factors. The cellline was named as TLR5-NFkB-SEAP. The reporter gene encodes a secretedenzyme, called embryonic alkaline phosphatase or SEAP. The SEAP reporteris placed under the control of the interferon-β minimal promoter fusedto five NFkB and AP-1-binding sites. The recombinant TLR5 binds to itsligand, or distinct pathogen-associated molecule, and initiates a chainof responses leading to recruitment of NFkB and AP1 transcriptionfactors to the reporter gene promoter, which induce expression of SEAP.

Cell culture and treatment: 500,000 gene reporter cells were grown andmaintained in DMEM medium supplemented with 10% fetal calf serum in T75flasks for three days at 37° C., 5% CO₂, and 95% humidity. For treatmentwith flagellin, wells of a 96-well plate were seeded with 10,000cells/well in 100 μL of growth medium. The cells were incubated for 72hours at 37° C., 5% CO₂, 95% humidity until day 4 after cells wereseeded onto wells of a 96-well plate. On day 4, medium (100 μL) waschanged to DMEM medium without fetal calf serum. S. subtilis and S.aureus flagellin were added at a range of concentration from 0.97 ng to1 ng/ml. Samples were taken at 6 and 24 hours later after addingflagellin Expression of reporter gene (SEAP) was quantified with acommercially available kit (SEAP Reporter Gene Assay of Cayman ChemicalCo., Ann Arbor, Mich.).

FIG. 11 shows both flagellins (B. subtilis and P. aeruginosa) couldactivate TLR5 downstream signaling pathways. Flagellin from B. subtilisstimulated TLR5-SEAP signaling transduction in a dose-dependent manner.The EC50 of flagellin on TLR5 was 0.02 ng/ml at 5 hours and 0.014 ng/mlat 23 hours. The EC50 of flagellin from Pseudomonas aeruginosa on TLR5was 0.0049 at 5 hours and 0.0019 at 23 hours. Flagellin from P.aeruginosa was more potent on stimulating TLR5 signaling pathways.

Example 11 Cell Culture and Virulence Determination of LPS fromDifferent Bacteria in THP1 Dual Reporter Cells

Bacterial cell wall and membrane components are recognized by TLR2. TLR2recognizes the microbial motifs PGN (peptidoglycan)/lipoproteins/dectinand LPS. TLR1 and TLR6 form heterodimers with TLR2 and bind totriacylated lipoproteins and diacylated lipoproteins, respectively. THP1NFkB-SEAP and IRF-Lucia™ Reporter Monocytes were purchased fromInvivogen, San Diego, Calif. THP1-Dual cells were derived from the humanTHP-1 monocyte cell line by stable integration of two inducible reporterconstructs. THP1-Dual cells feature the Lucia gene under the control ofan ISG54 (interferon-stimulated gene) minimal promoter in conjunctionwith five interferon-stimulated response elements. THP1-Dual cells alsoexpress a SEAP reporter gene driven by an IFN-b minimal promoter fusedto five copies of the NF-kB consensus transcriptional response elementand three copies of the c-Rel binding site. As a result, THP1-Dual cellsallow the simultaneous study of the NFkB pathway, by monitoring theactivity of SEAP, and the interferon regulatory factor (IRF) pathway, byassessing the activity of Lucia (IRF-Luc). Both reporter proteins arereadily measurable in the cell culture supernatant. This THP-1 cell linepossesses functional TLR1, TLR2, TLR4, TLR5, TLR6 and TLR8, purchasedfrom Invivogen. TLR4 senses LPS from Gram-negative bacteria while TLR5recognizes bacterial flagellin from both Gram-positive and Gram-negativebacteria, TLR8 detects long single-stranded RNA.

Culture and treatment: The THP1-dual cells were cultured in 15 ml growthmedium (RPMI 1640 with 10% heat-inactivated fetal bovine serum) in a T75flask at 37° C. and 5% CO₂. Cells were passed every 3 to 4 days byinoculating 300,000-500,000 cells/ml into a fresh T75 flask with 15 mlof fresh growth medium. To determine the effect of bacterial componentson reporter gene expression, wells in 96-well plates were seeded at100,000 cells in 90 μl of growth medium. 10 μl of bacterial wall andmembrane components, or heat-killed whole bacteria, were added to eachwell. After incubation for 18 hours at 37° C. and 5% CO₂, secretedluciferase and SEAP were quantified with commercially available assaykits (QUANTI-Luc of Invivogen, San Diego, Calif. for luciferase; SEAPReporter Gene Assay of Cayman Chemical Co., Ann Arbor, Mich. for SEAP).

DHP1-dual reporter cells were treated with three different preparationsof LPS as shown in FIG. 12A. All three LPS (ng/ml) activated productionof NFkB-SEAP reporter genes in a dose-dependent manner. In addition, Pg1690 LPS and E. coli LPS also stimulated expression of theIRF-luciferase reporter gene. TLR4 ligands, upon binding to TLR4receptors, activate at least two signaling pathways. One is a commonpathway NFkB-SEAP, which can be activated by all TLR ligands uponbinding to their specific receptors. For example, TLR2 ligand, LTA, canbind to TLR2 receptors and activate the NFkB-SEAP signaling pathway.Similarly, TLR4 ligand, LPS, upon binding to TLR4 receptors, also isable to activate the NFkB-SEAP signaling transduction. As shown in FIG.12A, E. coli LPS is a more potent ligand than P. gingivalis 1690 LPS onactivation of both NFkB-SEAP and IRF-luciferase signaling transduction.THP-1 cells produce several functional TLR receptors. And all TLRreceptors can activate the NFkB pathway, thus promoting expression ofthe NFkB-SEAP reporter gene. The reading of NFkB-SEAP is the collectiveactions of all TLR receptors, such as TLR2, TLR1, TLR6 and TLR4. All LPSfrom different bacteria stimulated NFkB-SEAP reporter gene.IRF-luciferase reporter gene, on the other hand, is driven by a limitednumber of TLR receptors, primarily TLR3, TLR4, TLR7, TLR8 and TLR9. BothP. gingivalis LPS 1690 and E. coli LPS stimulated expression ofIRF-luciferase in a dose-dependent fashion.

The THP-1 reporter cells were used to examine the clinical samples asdescribed for FIG. 7B of EXAMPLE 7. As shown in FIG. 12B, oral lavage(Healthy vs. Gingivitis) was evaluated using the IRF-Luc reporter genein THP-1 cells. After treatment with Crest® Pro-Health Clinical GumProtection Toothpaste (0.454% stannous fluoride) and Oral-B® IndicatorSoft Manual Toothbrush for four weeks, the virulence was reduced at week4 in both high and lower bleeders groups.

The THP-1 reporter cells were used to examine the clinical samples asdescribed for FIG. 7A of EXAMPLE 7. As shown in FIG. 12C, thesubgingival Plaque (Healthy vs. Gingivitis) was examined using the NFkBreporter gene in THP-1 cells. The baseline samples of the high bleedersgroup had higher RLU than those of the low bleeders. After treatmentwith Crest® Pro-Health Clinical Gum Protection Toothpaste (0.454%stannous fluoride) and Oral-B® Indicator Soft Manual Toothbrush for fourweeks, the virulence was reduced at week 4 in the bleeding sites in bothhigh and lower bleeders groups.

Example 12 Cell Culture and Virulence Determination of LTA fromDifferent Bacteria and Biofilms in THP1 Dual Reporter Cells

THP1 dual reporter cells also express TLR2, TLR1 and TLR6 receptors.Bacterial cell wall and some membrane components are recognized by TLR2,TLR1 and TLR6. TLR2 recognizes the microbial motifs PGN(peptidoglycan)/lipoproteins/dectin and LPS. To determine whether LTAfrom different bacteria have different effects on stimulating NFkB-SEAPreporter gene expression in the THP1 dual reporter cells, the cells wereprepared and treated in the same procedures as described in EXAMPLE 11.As shown in FIG. 13, LTA from both B. subtilis and S. aureus had similarpotency in promoting reporter gene expression in the THP1 dual reportercells.

Example 13 Distinguishing Bacterial Components from Different Bacteriain Human Primary Gingival Epithelial Cells

Primary human gingival epithelial cells were purchased from Zen-bio(Research Triangle Park, N.C.). and maintained in 15 ml of growth medium(CellnTec medium supplemented with CellnTec Growth Supplement, purchasedfrom CellnTec Advanced Cell Systems AG, Bern, Switzerland) in T75 flasksat 37° C. under a 5% CO2 atmosphere. As the experiment was done at 24 h,48 h and 72 h time point and with assay media, CellnTec medium alone orwith supplements, six 96 well plates were seeded with 7,500 cells/wellin 100 μl of CELLnTEC growth medium at 37° C. under a 5% CO2 atmosphere.The growth medium was changed to assay medium right before adding LPS orbacterial DNA. For example, if the assay medium was CellnTec mediumwithout supplements, 100 μl of CellnTec medium was added withoutsupplements in each well.

If the assay medium was the growth medium, 100 μl of growth medium wasadded to each well. The P. gingivalis LPS and bacterial DNA were addedto the cells. At 24 h, 48 h and 72 h, medium was collected for analysis.Cytokines were measured using Elisa kits from Meso Scale Discovery(Rockville, Md.), as per the manufacturer's instructions.

As shown in TABLE 4, human primary gingival epithelial cells weretreated with P. gingivalis DNA at 0, 0.3, 1 and 2 μg/ml. The cultureswere harvested at 24 and 48 hours after treatment and sixproinflammatory cytokines (interferon-γ, IL-1β, IL-2, IL-10, IL-12p70and TNF-α) were analyzed using ELISA kits from Meso Scale Discovery. Asshown in TABLE 4, expression of interferon-γ, IL-1β, IL-2, IL-10,IL-12p70 and TNF-α was low, or almost undetectable (each value is themean of three replicate in one experiment). They were not viablebiomarkers in distinguishing bacterial DNA. The results in TABLE 4illustrate the lack of inflammatory response from human gingivalepithelial cells, thus demonstrating the need to use an engineered cell.

TABLE 4 Screening of Human Gingival Epithelial Cells for InflammatoryMarkers treated with P. gingivalis DNA Production of cytokines (pg/ml)in primary human gingival epithelial cells treated with P. gingivalisgenomic DNA P. gingivalis P. gingivalis genomic 24 48 genomic 24 48 DNAhours hours DNA hours hours Interferon   0 ug 0 0.25 IL-1   0 ug 0 0.5gamma 0.3 ug 0 0.33 beta 0.3 ug 0 0.33   1 ug 0 0   1 ug 0 0   2 ug 0.330.67   2 ug 0 0 IL-10 PG DNA 0 ug 0.5 1 IL- PG DNA 0 ug 0 0.25 12p70 PGDNA 0.3 ug 0.67 0.33 PG DNA 0.3 ug 0.67 0 PG DNA 1 ug 0 0 PG DNA 1 ug 00.67 PG DNA 2 ug 0.33 0.33 PG DNA 2 ug 0 0.33 IL-2 PG DNA 0 ug 0.5 1TNF- PG DNA 0 ug 1 1.5 alpha PG DNA 0.3 ug 0 0.67 PG DNA 0.3 ug 1.671.33 PG DNA 1 ug 0.33 1 PG DNA 1 ug 0.67 1 PG DNA 2 ug 0.33 0.67 PG DNA2 ug 1.33 1.33

Human primary gingival epithelial cells were treated with P. gingivalisLPS at 0, 0.3, 1 and 2 μg/ml in the procedures described above. As shownin TABLE 5, again, expression of interferon-γ, IL-1β, IL-2, IL-10,IL-12p70 and TNF-α was low, or almost undetectable. They were not viablebiomarkers in distinguishing bacterial LPS in primary human gingivalepithelial cells. The results in TABLE 5 further illustrate how humangingival cells were not sufficiently sensitive to bacterial virulencefactors for an assay, and thus the need to utilize a cell line capableof demonstrating a dose dependent response to virulence factors.

TABLE 5 Screening of Human Gingival Epithelial Cells for InflammatoryMarkers treated with P. gingivalis LPS Production of cytokines (pg/ml)in primary human gingival epithelial cells treated with P. gingivalisLPS 24 h 48 h 24 h 48 h Interferon PG lPS 0 ug 0.00 0.25 IL-1 PG lPS 0ug 0.00 0.50 gamma PG LPS 0.3 ug 0.00 0.00 beta PG LPS 0.3 ug 0.00 0.67PG LPS 1 ug 0.33 0.33 PG LPS 1 ug 0.00 0.00 PG LPS 2 ug 0.00 0.00 PG LPS2 ug 0.00 1.00 IL-10 PG 1PS 0 ug 0.50 1.00 IL- PG lPS 0 ug 0.00 0.25 PGLPS 0.3 ug 0.67 0.67 12p70 PG LPS 0.3 ug 0.00 0.00 PG LPS 1 ug 0.00 0.67PG LPS 1 ug 0.00 0.33 PG LPS 2 ug 0.33 0.67 PG LPS 2 ug 0.67 0.00 IL-2PG lPS 0 ug 0.50 1.00 TNF- PG lPS 0 ug 1.00 1.50 PG LPS 0.3 ug 0.33 0.67alpha PG LPS 0.3 ug 1.33 0.67 PG LPS 1 ug 0.33 1.00 PG LPS 1 ug 2.001.33 PG LPS 2 ug 1.33 1.00 PG LPS 2 ug 0.67 1.33

Example 14 Gingivitis-Enriched Bacteria Reduced in Abundance in SixWeeks of Gingivitis Treatment

A randomized, two-group clinical study was conducted with 69 panelists(35 in the negative control group and 34 in the test regimen group).Panelists were 39 years old on average, ranging from 20 to 69, and 46%of the panelists were female. Treatment groups were well balanced, sincethere were no statistically significant (p>0.395) differences fordemographic characteristics (age, ethnicity, gender) or startingmeasurements for Gingival Bleeding Index (GBI); mean=29.957 with atleast 20 bleeding sites, and Modified Gingival Index (MGI); mean=2.086.All sixty-nine panelists attended each visit and completed the treatmentprocess. The following treatment groups were compared over a 6-weekperiod:

Test regimen: Crest® Pro-Health Clinical Plaque Control (0.454% stannousfluoride) dentifrice, Oral-B® Professional Care 1000 with PrecisionClean brush head and Crest® Pro-Health Refreshing Clean Mint (0.07% CPC)mouth rinse. Control regimen: Crest® Cavity Protection (0.243% sodiumfluoride) dentifrice and Oral-B® Indicator Soft Manual toothbrush.

Dental plaques were collected from the same panelists in the testregimen in the clinical study as described in EXAMPLE 2. A supragingivalsample was taken from each panelist with a sterile curette at thetooth/gum interface, using care to avoid contact with the oral softtissue. Plaques were sampled from all available natural teeth (upperarch only) until no plaque was visible. Following sampling, the plaquesamples were placed into a pre-labeled (panelist ID, sample initials,visit, and date) Eppendorf tube with 1 ml of PBS/Glycerol buffer andabout 50 of sterile 1 mm glass beads, stored on ice until all sampleswere collected. The samples were then transferred to a −70° C. freezerfor storage until further processing. Genomic DNA was isolated fromsupragingival plaque samples using QIAamp® genomic DNA kits (Qiagen,Germany) following manufacturer's instruction. Metasequencing wascarried out at BGI Americas Corporation (Cambridge, Mass.). All datawere analyzed at Global Biotech of Procter & Gamble Company in Mason,Ohio.

Clinical measurements: Bleeding sites (GBI) were decreased in the testregimen significantly on week 1, 3 and 6 in comparison to the controlregimen (FIG. 14). Similarly, Inflammation (MGI) grades also decreasedin the test regimen (FIG. 14).

Genomic DNA of the supragingival plaques in the test regimen wassequenced. As shown in FIG. 15, abundance of certain bacteria in thesupragingival plaques changed in the six week treatments. Certainbacteria, such as Porphyromonas sp oral taxon 279 and Prevotellapallens, were decreased in weeks 1 and 3 (FIG. 15). The amount of eachbacterial species was plotted over the four time periods of thetreatment. The amount of certain bacteria, such as Peptostreptococcusstomatis and Prevotella intermedia, was reduced during the six week oftreatment as shown in FIG. 15.

Example 15 Production of Cytokines, Chemokines and Other BioactiveProteins Decreased as Gingivitis Symptoms were Alleviated During 6 Weekof Treatment

In the same clinical study as described in EXAMPLE 14, gingival-brushsamples were collected from the same panelists as in EXAMPLE 14. Beforesampling, panelists rinsed their mouths for 30 seconds with water. Adental hygienist then sampled the area just above the gumline using abuccal swab brush (Epicentre Biotechnologies cat.# MB 100SP). The swabwas immediately placed into 1 ml extraction buffer [PBS, 0.25M NaCl, 1×Halt™ Protease Inhibitor Single-Use Cocktail (Lifetechnologies, GrandIsland, N.Y.)] in a 1.5 ml Eppendorf tube vortexed for 30 seconds, andimmediately frozen on dry ice and stored in a −80 C freezer untilanalysis. The samples were taken out of freezer, thawed and extracted byplacing the samples on a tube shaker for 30 minutes at 4° C. The tubeswere centrifuged at 15000 RPM for 10 min in Eppendorf Centrifuge 5417R(Eppendorf, Ontario, Canada) to pellet any debris. The extract (800 μl)was analyzed for protein concentrations using the Bio-Rad protein assay(BioRad, Hercules, Calif.).

Forty proteins were measured in the gingival samples using V-PLEX HumanBiomarker 40-Plex Kit (Meso Scale Diagnostics Rockville, Md.). The assaywas performed following the manufacturer's instruction.

Among the proteins measured in the gingival samples, most proteins inthe Proinflammatory Panel 1 (human), Cytokine Panel 1 (human), ChemokinePanel 1 (human), Angiogenesis Panel 1 (human), and Vascular Injury Panel2 (human) had significant changes in their abundance during the 6-weektreatment (TABLE 6). Those include FN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8,IL-10, IL-12p70, IL-13, TNF-α, GM-CSF, IL-5, IL-16, IL-7,IL-12/IL-23p40, IL-1α, VEGF-A, IL-17A, IL-15, TNF-β, IL-8 (HA), MCP-1,MCP-4, Eotaxin, IP-10, MDC, Eotaxin-3, TARC, MIP-1α, MIP-1β, VEGF-C,VEGF-D, Tie-2, Flt-1/VEGFR1, PlGF, FGF (basic), SAA, CRP, VCAM-1, andICAM-1.

TABLE 6 Changes in abundance of proteins in the gingival-brush samplesMean α = 0.05 Baseline Week 1 Week 3 Week 6 Baseline Week 1 Week 3 Week6 ICAM-1 16.035 12.209 10.090 9.767 A B B, C C IL-1α 3.554 2.331 2.1811.891 A A, B B, C C IL-1β 53.666 35.575 24.295 24.440 A B C C TNF-β0.0013 0.0010 0.0008 0.0007 A B C C IL-12p70 0.172 0.148 0.118 0.127 AA, B C B, C IL-13 0.805 0.762 0.624 0.648 A A, B C B, C IL-4 0.127 0.1150.090 0.096 A A, B C B, C IL-5 0.004 0.003 0.002 0.003 A B C B, C CRP15.637 12.743 12.385 5.809 A A A B Eotaxin 0.077 0.064 0.059 0.059 A A,B B B GM-CSF 0.010 0.008 0.008 0.008 A B B B IFNγ 0.530 0.446 0.3780.386 A A, B B B IL-10 0.875 0.490 0.423 0.244 A A, B B B IL-15 0.0050.003 0.003 0.003 A B B B IL-16 0.466 0.345 0.342 0.295 A B B B IL-60.196 0.192 0.168 0.150 A A A, B B IL-7 0.004 0.003 0.003 0.003 A B B BIL-8 856.276 652.066 567.361 572.602 A B B B MCP-1 0.053 0.047 0.0390.039 A A, B B B MDC 0.399 0.407 0.345 0.339 A A B B SAA 7.039 6.9056.092 5.162 A A A, B B Tie-2 0.273 0.239 0.267 0.221 A A, B A B VCAM-14.971 3.706 3.156 2.892 A B B B VEGF 0.625 0.511 0.478 0.480 A B B BVEGF 2 0.772 0.661 0.620 0.582 A B B B VEGF-D 0.057 0.052 0.051 0.045 AA, B A, B B VEGF-C 0.145 0.149 0.125 0.137 A, B A B A, B TARC 0.0200.029 0.019 0.019 A B A A bFGF 0.020 0.015 0.012 0.013 A A A A Eotaxin-30.095 0.108 0.091 0.094 A A A A Flt-1 0.390 0.518 0.433 0.415 A B A, B AIL-12p40 0.039 0.031 0.028 0.031 A A A A IL-2 0.166 0.199 0.210 0.162 AA A A IL-8 (HA) 47.508 44.362 41.260 39.119 A A A A IP-10 0.540 1.6880.740 0.606 A A A A MCP-4 0.023 0.023 0.020 0.022 A A A A MIP-1α 0.0910.091 0.084 0.080 A A A A MIP-1 0.091 0.100 0.110 0.094 A A A A TNFα2.009 2.067 2.021 1.670 A A A A

Example 16 One Hundred Seventy Metabolites were Identified in GingivalSamples

The same gingival-brush samples as described in EXAMPLE 15 were used formetabonomic analyses. Fourteen panelists were selected randomly fromeach treatment group to determine if any metabolite concentrations werechanged in gingival samples during the first 3 weeks of treatment. Bothbaseline and week 3 samples were sent to Metabolon, Inc. (Durham, N.C.)for metabonomic measurement. 170 metabolites were identified andquantified. As shown in TABLE 7, some metabolite concentrations werechanged during the first 3 weeks of treatment. Citrulline concentrationsin the gingival samples were reduced after three weeks of treatment inthe treatment regimen group. Similarly, ornithine was also reduced inthe treatment regimen group after three weeks of treatment. Reduction ofcitrulline and ornithine was likely associated with alleviation ofgingivitis.

TABLE 7 Comparison of metabolites in gingival brush samples betweenbaseline and week 3 during gingivitis treatment Baseline 3 week 3 week/Biochemical Name mean mean baseline p-value q-value Mass 13-HODE +9-HODE 1.0877 0.7088 0.65 0.0601 0.1338 295.2 1-arachidonoylglycero-1.2294 0.8274 0.67 0.038 0.1035 500.3 phosphoethanolamine1-oleoylglycero- 0.7378 1.0747 1.46 0.0767 0.1548 478.3phosphoethanolamine 2-methylbutyrylcarnitine 1.7769 0.6997 0.39 0.00340.0546 246.1 (C5) adenosine 5′-monophosphate 1.4092 0.8451 0.6 0.02950.0956 348.1 (AMP) alanine 0.8721 1.102 1.26 0.0318 0.0973 115.9arginylleucine 1.4447 0.6819 0.47 0.0084 0.0777 288.3arginylphenylalanine 0.9616 0.3335 0.35 0.0119 0.0777 322.2asparagylleucine 0.9295 0.6122 0.66 0.0698 0.1465 246.2 citrulline1.0147 0.71 0.7 0.0104 0.0777 176.1 deoxycarnitine 3.2381 0.6088 0.190.0003 0.0168 146.1 EDTA 1.5985 0.8384 0.52 0.0138 0.0777 291.1erythritol 1.625 0.8085 0.5 0.0582 0.1325 217 fructose 1.9933 1.11060.56 0.0847 0.1605 217 glutamine 1.2459 0.8366 0.67 0.0374 0.1035 147.2glutathione, oxidized (GSSG) 1.0161 1.4669 1.44 0.087 0.1605 613.1glycerol 1.3783 0.8308 0.6 0.0391 0.1035 205 lauryl sulfate 1.685 0.86230.51 0.0397 0.1035 265.2 leucine 1.2158 0.9359 0.77 0.0613 0.1338 132.2leucylleucine 0.9505 0.4393 0.46 0.0251 0.0877 245.1 lysylleucine 1.20090.5275 0.44 0.0036 0.0546 260.2 lysylphenylalanine 1.1682 0.4563 0.390.0095 0.0777 294.3 maltose 0.8727 1.4481 1.66 0.022 0.0877 204.1maltotriose 1.0456 1.8347 1.75 0.0858 0.1605 204 mannitol 1.3004 0.79820.61 0.042 0.107 319.1 ornithine 1.2916 0.7069 0.55 0.0367 0.1035 141.9palatinitol 1.4395 0.8272 0.57 0.0782 0.1549 204 phosphate 1.4008 0.83760.6 0.0208 0.0877 298.9 proline 1.405 0.99 0.7 0.0033 0.0546 116.1propionylcarnitine 1.2565 0.7688 0.61 0.0201 0.0877 218.2 pyroglutamine1.3424 0.7873 0.59 0.0136 0.0777 129.2 serylisoleucine 1.1753 0.71690.61 0.0814 0.1583 219.2 spermidine 1.1613 0.8678 0.75 0.0687 0.1465146.2 succinate 1.2929 0.8113 0.63 0.0754 0.1548 247 threonylleucine1.1513 0.4931 0.43 0.0044 0.0594 231.2 threonylphenylalanine 1.76930.918 0.52 0.0233 0.0877 267.2 trehalose 2.3563 0.9084 0.39 0.00540.0647 361.2 tryptophan 1.1518 0.9089 0.79 0.0487 0.1185 205.1 tyrosine1.383 1.0299 0.74 0.0161 0.0787 182.1 valine 1.1598 0.9271 0.8 0.03040.0956 118.1 valylvaline 0.9347 0.8231 0.88 0.0508 0.1207 215.2 X-136710.5035 0.918 1.82 0.0545 0.1267 315.3 X-14588 1.3647 0.8378 0.61 0.0240.0877 151 X-16103 1.3643 0.8461 0.62 0.0297 0.0956 99.3 X-17266 1.31580.576 0.44 0.0003 0.0168 530.4 X-17375 1.4785 0.8387 0.57 0.0189 0.0877357.1 X-18472 0.6138 1.1441 1.86 0.0011 0.0405 827.1 X-18779 1.37560.8035 0.58 0.0162 0.0787 209.1 X-19607 1.5237 0.7167 0.47 0.002 0.0537366.1 X-19609 1.3284 0.7721 0.58 0.016 0.0787 204 X-19612 1.3896 0.78430.56 0.01 0.0777 427.2 X-19613 1.3412 0.7535 0.56 0.0099 0.0777 429.3X-19614 1.3378 0.7343 0.55 0.0454 0.113 570.1 X-19807 1.3478 0.8411 0.620.0244 0.0877 93 X-19808 1.3348 0.8368 0.63 0.0254 0.0877 95 X-198501.3576 0.7519 0.55 0.011 0.0777 334.2 X-19857 1.3357 0.8032 0.6 0.0380.1035 230 X-20000 1.2784 0.7536 0.59 0.0133 0.0777 81.2

Example 17 Citrulline was Reduced in Regimen Treatment Over 6 Weeks

Quantitation of citrulline and ornithine from the extracts of theGingival-brush samples was conducted using gradient hydrophilicinteraction liquid chromatography with tandem mass spectrometry(HILIC/MS/MS). Gingival-brush samples were obtained from the same humanpanelists in the clinical study as described in EXAMPLE 14, and wereplaced into extraction buffer as described in EXAMPLE 15. Thesupernatants were subject to both HILIC/MS/MS and BCA analysis. For freecitrulline and ornithine analysis, the extracts of the Gingival-brushsamples were analyzed either directly (50 μl undiluted sample solution)in 50/50 acetonitrile/ultra-pure water with 0.754% formic acid ordiluted fivefold. For total citrulline and ornithine analysis, theextracts of the Gingival-brush samples were first hydrolyzed using 6 NHCl (50 μL of extract with 450 μL of 6N HCl), no shaking, and placed ona hot plate at 110° C. for 16 hours. The hydrolyzed samples were thendried down under vacuum at room temperature (Savant speedvac ofLifetechnology, Grand Island, N.Y.) and then reconstituted in 1 ml ofdilution solution (50/50 acetonitrile/ultra-pure water with 0.754%formic acid) for analysis. The standards and the samples were analyzedusing gradient hydrophilic interaction liquid chromatography with tandemmass spectrometry (HILIC/MS/MS). Analytes and the corresponding ISTDs(stable isotope labeled internal standard) were monitored byelectrospray ionization (ESI) in positive mode using theselected-reaction-monitoring schemes shown in TABLE 8. A standard curvewas constructed by plotting the signal, defined here as the peak arearatio (peak area analyte/peak area ISTD), for each standard versus themass of each analyte for the corresponding standard. The mass of eachanalyte in the calibration standards and Gingival-brush extract sampleswere then back-calculated using the generated regression equation. Theconcentration of protein bound citrulline or ornithine was calculated asthe result of subtracting the concentration of free citrulline orornithine from the concentration of total citrulline or ornithine,respectively. The result was reported as the concentration of citrullineor ornithine or the result was standardized by dividing by the amount ofcitrulline or ornithine by the amount of the total proteins that werefound in the extract.

TABLE 8 Multiple Reaction Monitoring (MRM) transitions for analytes andtheir corresponding stable isotope labeled internal standards AnalytesMRM Internal Standards MRM Citrulline 176 → 159 d₇-Citrulline 181 → 164Ornithine 133 → 70  d₆-Ornithine 139 → 76 

All samples from all panelists of the Test regimen [Crest® Pro-HealthClinical Plaque Control (0.454% stannous fluoride) dentifrice, Oral-B®Professional Care 1000 with Precision Clean brush head and Crest®Pro-Health Refreshing Clean Mint (0.07% CPC) mouth rinse] were analyzed.As shown in FIG. 16, citrulline levels reduced rapidly in the first weekof treatment, and then continued to decline gradually in weeks 3 and 6of treatment. These results are consistent with clinical observations,where gingival bleeding sites (GBI) and the gingival inflammation (MGI)were reduced over the 6-week treatment period.

Example 18 Levels of Proteins Containing Ornithine Decreased Over 6 WeekTreatment

The same samples as described in EXAMPLE 17 were analyzed usingprocedures as described in EXAMPLE 17. Gingivitis was treated for 6weeks. Baseline (BL) represents diseased status. Symptoms of gingivitiswere alleviated from week 1 to week 6 treatments. Protein boundornithine (the difference between total and the free ornithine) washigher in gingivitis as shown in FIG. 17. Protein bound ornithine wasreduced gradually as gingivitis was decreased in severity.

Example 19 Expression of Enzymes in the Ornithine-Citrulline-ArginineCycle was Changed in Gingival Samples During the 6 Week Treatment

Gingival samples were collected as described in EXAMPLES 15, from thesame panelists as in EXAMPLE 15, and were used to examine the expressionof genes during 6 weeks of treatments with Test regimen [Crest®Pro-Health Clinical Plaque Control (0.454% stannous fluoride)dentifrice, Oral-B® Professional Care 1000 with Precision Clean brushhead and Crest® Pro-Health Refreshing Clean Mint (0.07% CPC) mouthrinse] and Control regimen [Crest® Cavity Protection (0.243% sodiumfluoride) dentifrice and Oral-B® Indicator Soft Manual toothbrush].

After harvesting the samples, the brush was completely immersed in theRNAlater solution (1 ml in in a 1.5 ml Eppendorf tube) for keeping RNAfrom degrading during transport and storage (Qiagen, Valencia, Calif.).The microcentrifuge tubes were vortexed/mixed for 30 seconds,immediately frozen on dry ice, stored and transferred on dry ice to thelab for biomarker analysis. RNA isolation and microarray analysis wereperformed as described previously in a publication (Offenbacher S,Barros S P, Paquette D W, Winston J L, Biesbrock A R, Thomason R G, GibbR D, Fulmer A W, Tiesman J P, Juhlin K D, Wang S L, Reichling T D, ChenK S, Ho B. J Periodontol. 2009 December; 80(12): 1963-82. doi:10.1902/jop.2009.080645. Gingival transcriptome patterns duringinduction and resolution of experimental gingivitis in humans).

The ornithine-citrulline-arginine cycle consists of four enzymes (FIG.18). The main feature of the cycle is that three amino acids (arginine,ornithine, and citrulline) can be converted to each other. The firstenzyme is ornithine transcarbamoylase, which transfers a carbamoyl groupfrom carbamoyl phosphate to ornithine to generate citrulline. Thisreaction occurs in the matrix of the mitochondria. Expression ofornithine transcarbamoylase was reduced in the treatment (FIG. 19).

The second enzyme is argininosuccinate synthetase. This enzyme uses ATPto activate citrulline by forming a citrullyl-AMP intermediate, which isattacked by the amino group of an aspartate residue to generateargininosuccinate. This and subsequent two reactions occur in thecytosol. Again, expression of argininosuccinate synthetase decreasedduring the treatment. The third enzyme is argininosuccinate lyase, whichcatalyzes cleavage of argininosuccinate into fumarate and arginine. Thelast enzyme is argininase. Argininases cleave arginine to produce ureaand ornithine. In a contrast to the decreased expression of ornithinetranscarbamoylase and argininosuccinate synthetase genes, argininase Iand II increased (FIG. 19).

Arginine is also a substrate for nitric oxide synthase, which oxidizesarginine to produce citrulline and nitric oxide. Expression of nitricoxide synthase gene increased too (FIG. 19).

Example 20 Citrulline Increased in Gingival Samples in ExperimentGingivitis

Experimental Gingivitis:

Another clinical study was carried out to determine whether citrullineis increased in experimentally induced gingivitis in healthy humanpanelists. This was a case-control study enrolling 60 panelists. Thestudy population included two groups as follows: Group 1 or highbleeders group, thirty (30) panelists with at least 20 bleeding sites,where bleeding is a GBI site score of 1 or 2 at baseline. Group 2 or lowbleeders group, thirty (30) panelists with 2 or less bleeding sites,where bleeding is a GBI site score of 1 or 2.

The Study Consisted of Two Phases:

Health/Rigorous Hygiene Phase with dental prophylaxis, polishing andrigorous oral hygiene; and Induced Gingivitis Phase without oralhygiene. At the Screening visit, panelists underwent an oral soft tissueassessment and had a gingivitis evaluation (Modified Gingival Index(MGI) and Gingival Bleeding Index (GBI). At Visit 2 qualifying panelistsreceived an oral soft tissue exam followed by a gingivitis evaluationand gingival plaques and gum swabs were collected for the qPCR, proteinand RNA host biomarker analysis. Following that, all panelists receiveddental prophylaxis and entered the Health/Rigorous Hygiene Phase,lasting two weeks. After two weeks of rigorous hygiene, panelistsentered the Induced Gingivitis Phase, lasting for three weeks. Oral softtissue exams and gingivitis were re-evaluated and all samples (gumswabs) were collected at Baseline, WK0 and WK2.

Gingival Sample Collection—

A gum swab was collected from each side of the upper arch using theprocedures as described in EXAMPLE 15. Gum swabs were collected close tothe gum line from the buccal sites only (preferably from four adjacentteeth—preferably from premolar and molar areas). Panelists rinsed for 30seconds with 15 ml of Listerine rinse to clean the surface of samplingarea. After the Listerine rinse, panelists rinsed for 30 seconds with 20ml of water. Following that, selected sites were isolated with a cottonroll and gently dried with an air syringe and two gum swabs were takenwith collection brushes/swabs from the gingiva region close to thegumline of the selected teeth. The samples were placed in a pre-labeled(panelist ID, sample ID, visit, and date) 1.5 ml micro-centrifuge tubecontaining 800 ul DPBS (Dulbecco's phosphate-buffered saline)(Lifetechnologies, Grand Island, N.Y.) with protease inhibitors,including AEBSF (4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride)2 mM, aprotinin 0.3 μM, Bestatin 130 μM, EDTA(Ethylenediaminetetraacetic acid) 1 mM, E-64 1 μM, and leupeptin 1 μM.The vials were vortexed/mixed for 30 seconds, immediately frozen on dryice, stored and transferred on dry ice to the lab for biomarkeranalysis. Samples from three visits were analyzed using the proceduresdescribed in EXAMPLE 17, and shown in FIG. 20. Those three visits werebaseline, Week 0, (right after the Health/Rigorous Hygiene Phase andbefore the induced gingivitis phase) and week 2 (at the end of InducedGingivitis Phase). Free citrulline levels were low in both the high andlow bleeders groups at the baseline and week 0, but rose quickly in theinduced gingivitis in both groups at week 2.

Example 21 Levels of Proteins Containing Citrulline Decreased inExperimentally Induced Gingivitis

The same procedures were used as described in EXAMPLE 17. The sampleswere the same as described in EXAMPLE 20. Protein bound citrulline waslower at the baseline than that at week 0 in both high and low bleedersgroups as shown in FIG. 21 in gingival tissue. It was low inexperimental gingivitis in both groups at week 2.

Example 22 Levels of Proteins Containing Ornithine Increased in GingivalSamples in Experiment Gingivitis

The same clinical samples from experimental gingivitis (EXAMPLE 20) wereanalyzed using the procedures described in EXAMPLE 17. The boundornithine was the lowest at week 0 (FIG. 22) in both groups. Its levelsat the baseline were higher than those at week 0. The bound ornithinereached peaks when gingivitis was induced in both groups at week 2. Alsoit is worth noting the total ornithine (Free and protein boundornithine) was increased in the induced gingivitis (FIG. 23) in bothgroups.

Example 23 Levels of Proteins Containing Arginine Decreased in GingivalSamples in Experimentally Induced Gingivitis

The same procedures were used as described in EXAMPLE 17. The sampleswere the same as described in EXAMPLE 20. The protein bound arginine wasthe lowest in induced gingivitis (FIG. 24) in both groups. Its levelswere higher in WK0 than at Baseline in both groups. The total argininein the gingival brush samples displayed the same patterns as the proteinbound one (FIG. 25).

Example 24 Citrulline Inhibited LPS Stimulated Production ofProinflammatory Cytokine in Human THP-1 Cells

Citrulline was purchased from Sigma-Aldrich (St. Louis, Mo.). THP1-Dual™cells were purchased from Invivogen (San Diego, Calif.). Cells werecultured following the manufacturer's instruction, as described inEXAMPLE 11. For treatment, 0.3 mM to 9 mM of citrulline were first addedto the culture medium. Then, 300 ng/ml of P. gingivalis LPS 1690 wereadded 60 minutes later. After 24 hours of treatment, media was collectedand analyzed for cytokine production using 9-plex kit (Meso ScaleDiagnostics Rockville, Md.).

P. gingivalis LPS 1690 stimulated cytokine production, as shown in FIG.26. Citrulline inhibited P. gingivalis LPS 1690 effects onproinflammatory cytokine production in a dose-dependent manner. Thosecytokines include IL-6, TNF-α, IL-12p70, IL-10, IL-2, IFN-r and IL-1β.

Example 25 Binding of BODIPY-TR-Cadaverine to Bacteria and theirProducts

Bacteria and their products can activate TLR2 and TLR4 reporter genes asdescribed in EXAMPLES 5 and 8. Here, experiments were carried out todetermine whether different bacteria and their products have differentIC50 in reducing fluorescent intensity of BODIPY-TR-cadaverine,hereinafter referred to as BC, in a procedure as described in EXAMPLE 4.Briefly, high throughput screen utilizes the fluorescent dyeBODIPY-TR-cadaverine5-(((4-(4,4-difluoro-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino)pentylaminehydrochloride (BC fluorescent dye), purchased from Life Technologies(Carlsbad, Calif.), as described previously by Wood, Miller and David(2004) (Comb Chem High Throughput Screen 2004 May; 7(3):239-49.Anti-endotoxin agents. 1. Development of a fluorescent probedisplacement method optimized for the rapid identification oflipopolysaccharide-binding agents.). The experiment was carried out atroom temperature. E. coli LPS (15 μg/ml) was mixed in a well of a96-well solid black flat bottom plate (Corning Incorporated, Corning,N.Y.) with SnF2 and cetylpyridinium chloride in 30 μl of 50 mM Trisbuffer at pH 7.4 for 10 mM, and then 20 μl of 60 μM BC fluorescent dyewas added right before fluorescence measurement at 21° C., or roomtemperature. Fluorescent measurements were performed in a SpectraMax M3automated 96 well plate reader (Molecular Device, Sunnyvale, Calif.).The excitation wavelength was 580 nM and the fluorescence emission wasmeasured at 620 nM. The half maximal inhibitory concentration (IC₅₀) isa measure of the effectiveness of bacterial products inhibiting BCemitting fluorescence. It indicates how much of a bacterial material isneeded to inhibit BC fluorescence by half using GraphPad Prism software(GraphPad Software, La Jolla, Calif.). As shown in FIGS. 27 and 28, eachbacterial product decreases the fluorescent intensity of BC in adose-dependent manner. Similarly, the whole bacterial cells and thesupernatant of bacterial growth also reduced the fluorescence of BC in adose-dependent fashion (FIGS. 29 and 30).

Example 26 Determination of Bacterial Virulence

Growth of bacteria: Two bacteria, Bacterium A and Bacterium B, werecultured in Tryptic Soy Broth medium (Sigma-Aldrich, St. Louis, Mo.) at37° C. with shaking at 200 rpm. The bacteria were harvested at 24 hours,and suspended in 0.5 ml of phosphate-buffered saline, labeled “live”.Half ml of “Live” bacteria was transferred to a 1.5 ml microtube, andheated to 80° C. for 30 min. The heat-treated bacteria were labeled“Heat-Inactivated”, or “Dead”.

Measurement of TLR responses in THP-1 gene reporter cells (NFkB-SEAP):The Live and Heat-Inactivated bacteria were applied to THP-1 cells asdescribed in EXAMPLE 11. As shown in FIG. 31, EC50 of Bacterium A and Bon activation of NFkB-SEAP reporter gene in THP-1 cells was determined.Both Live and Heat-inactivated (Dead) bacteria stimulated expression ofthe NFkB-SEAP reporter gene. Bacterium B had a lower EC50 than BacteriumA in activating expression of the NFkB-SEAP reporter gene.

Cytokine production and measurement: Human peripheral bleed mononuclearcells (hPBMC) were obtained from All Cells company (All Cells, Alameda,Calif.) as Leukapheresed blood.

Leukapheresed blood was mixed with an equal part of DMEM+glutaGROsupplemented with 9.1% fetal bovine serum and 1% penicillin/streptomycin(Thermo Fisher, Waltham, Mass.). hPBMC were isolated from the 1:1mixture of blood and culture medium by collecting the buffy coat of acentrifuged Histopaque®-1077 (Sigma-Aldrich, St. Louis, Mo.) bufferdensity gradient. The cells (200,000 cells) were cultured in 200 μl ofDMEM+glutaGRO supplemented with 9.1% fetal bovine serum and 1%penicillin/streptomycin, and treated with Live and Heat-Inactivebacteria (6,250,000 colony-forming units). The medium was harvested at24 hours after adding the bacteria, and analyzed for proinflammatorycytokines in a kit following manufacturer's instruction (Meso ScaleDiagnostics, Rockville, Md.).

As shown in TABLE 9, both live bacterium A and B stimulated productionof cytokines in hPBMC. Bacteriun B was far more potent than Bacterium Ain promoting production of IFN-T, IL-10, IL-12p70, IL-1β, IL-6, IL-8 andTNF-α in hPBMC.

Statistics Cytokines Live Bacterium A Live Bacterium Mean IFN-γ 867.1612734.36 Mean IL-10 68.35 412.51 Mean IL-12p70 23.75 253.47 Mean IL-1β2300.86 6969.31 Mean IL-2 44.24 65.17 Mean IL-6 3431.05 9963.84 MeanIL-8 65742.91 70357.25 Mean TNF-α 3710.66 13825.49 Std Dev IFN-γ 488.118200.13 Std Dev IL-10 33.56 314.23 Std Dev IL-12p70 14.71 283.33 Std DevIL-1β 1569.72 7691.93 Std Dev IL-2 32.97 36.29 Std Dev IL-6 2212.626552.12 Std Dev IL-8 18689.24 13669.28 Std Dev TNF-α 2503.25 8302.26

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “40 mm” is intended to mean“about 40 mm.”

Every document cited herein, including any cross referenced or relatedpatent or application and any patent application or patent to which thisapplication claims priority or benefit thereof, is hereby incorporatedherein by reference in its entirety unless expressly excluded orotherwise limited. The citation of any document is not an admission thatit is prior art with respect to any invention disclosed or claimedherein or that it alone, or in any combination with any other referenceor references, teaches, suggests or discloses any such invention.Further, to the extent that any meaning or definition of a term in thisdocument conflicts with any meaning or definition of the same term in adocument incorporated by reference, the meaning or definition assignedto that term in this document shall govern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

What is claimed is:
 1. A method of determining bacterial virulence in anoral cavity comprising: a. providing a reporter cell expressing at leastone toll-like receptor; b. providing a sample of oral matter; c.combining the sample of oral matter and the reporter cell; d. measuringthe toll-like receptor activation.
 2. The method of claim 1, wherein thetoll-like receptor is at least one of TLR1 to TLR9.
 3. The method ofclaim 1, wherein the reporter cell expresses at least of one TLR2 orTLR4.
 4. The method of claim 1, wherein the toll-like receptor activatesa reporter gene.
 5. The method of claim 4, wherein the reporter gene isa secreted embryonic alkaline phosphatase (SEAP) reporter gene.
 6. Themethod of claim 1, wherein the sample of oral matter is at least one ofgum-line plaque, subgingival plaque, supragingival plaque, interstitialplaque, gingival crevicular fluid (GCF), gingival biopsy, saliva, ortongue swab.
 7. The method of claim 1, wherein the sample of oral mattercomprises virulence factor that is at least one of Gram positive cellwall components, Gram negative cell wall components, bacterial DNA,flagellin, peptidoglycan, bacterial metabolites, fimbriae; outermembrane vesicles bacterial proteins, or bacterial enzymes.
 8. Themethod of claim 7, wherein the sample of oral matter comprises virulencefactor that is at least one of lipopolysaccharide or lipoteichoic acid.9. The method of claim 1 comprising the additional steps of: e.providing another sample of oral matter from an individual who used anoral care composition prior to providing another sample of oral matter;f. combining the another sample of oral matter and the reporter cell; g.measuring the toll-like receptor response; h. comparing the toll-likereceptor response from the sample of oral matter and another sample oforal matter.
 10. The method of claim 9, where the sample of oral matterand the another sample of oral matter are from the same individual. 11.A method for determining the virulence of lipopolysaccharide comprising:a. providing a lipopolysaccharide; b. providing a reporter cellexpressing at least one Toll-like receptor; c. combining the reportercell with the lipopolysaccharide; d. measuring the toll-like receptoractivation; e. quantifying the lipopolysaccharide.
 12. The method ofclaim 11, wherein the Toll-like receptor activation is determined bymeasuring at least one of EC50 or fold of stimulation.
 13. The method ofclaim 11, wherein the Toll-like receptor is at least one of TLR1 toTLR9.
 14. The method of claim 11, wherein the reporter cell expresses atleast one of TLR2 or TLR4.
 15. The method of claim 11, wherein theToll-like receptor activates a reporter gene.
 16. The method of claim15, wherein the reported gene is a secreted embryonic alkalinephosphatase (SEAP) reporter gene.
 17. The method of claim 11, whereinthe lipopolysaccharide is quantified using at least one of limulusamebocyte lysate assay or BODIPY TR cadaverine assay.
 18. A method fordetermining the virulence of lipoteichoic acid comprising: a. providinga lipoteichoic acid; b. providing a reporter cell expressing at leastone Toll-like receptor; c. combining the reporter cell with thelipoteichoic acid; d. measuring the toll-like receptor activation; e.quantifying the lipoteichoic acid.
 19. The method of claim 18, whereinthe Toll-like receptor activation is determined by measuring at leastone of EC50 or fold of stimulation.
 20. The method of claim 18, whereinthe Toll-like receptor is at least one of TLR1 to TLR9.